Monoclonal Antibodies That Specifically Block Biological Activity Of A Tumor Antigen

ABSTRACT

This invention relates to novel monoclonal antibodies that specifically bind to the alpha-folate receptor. In some embodiments, the antibodies inhibit a biological activity of folate receptor-α (FR-α). The antibodies are useful in the treatment of certain cancers, particularly cancers that have increased cell surface expression of the alpha-folate receptor (“FR-α”), such as ovarian, breast, renal, colorectal, lung, endometrial, or brain cancer. The invention also relates to cells expressing the monoclonal antibodies, antibody derivatives, such as chimeric and humanized monoclonal antibodies, antibody fragments, and methods of detecting and treating cancer using the antibodies, derivatives, and fragments.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.12/500,144, filed Jul. 9, 2009, which is a continuation of U.S.application Ser. No. 11/056,776, filed Feb. 11, 2005, which claimsbenefit of U.S. Appl. No. 60/544,364, filed Feb. 12, 2004. The contentof each of these applications is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to purified novel monoclonal antibodies thatspecifically bind to the alpha-folate receptor (“FR-α”) and compositionsthereof. In some embodiments, the antibodies of the invention block thebiological activity of FR-α. The antibodies and compositions of theinvention are useful in the treatment of certain cancers, particularlycancers that have increased cell surface expression of the alpha-folatereceptor, such as ovarian, breast, renal, colorectal, lung, endometrial,or brain cancer. The invention also relates to hybridoma cellsexpressing the monoclonal antibodies, antibody derivatives, such aschimeric and humanized monoclonal antibodies, antibody fragments,mammalian cells expressing the monoclonal antibodies, derivatives andfragments, compositions of purified antibodies of the invention, andmethods of detecting and treating cancer using the antibodies,derivatives, fragments, and compositions of the invention.

BACKGROUND OF THE INVENTION

There are three major isoforms of the human membrane folate bindingprotein, α, β, and γ. The α and β isoforms have about 70% amino acidsequence homology, and differ dramatically in their stereospecificityfor some folates. Both isoforms are expressed in fetal and adult tissue,although normal tissue generally expresses low to moderate amounts ofFR-β. FR-α, however, is expressed in normal epithelial cells, and isfrequently strikingly elevated in a variety of carcinomas (Ross et al.(1994) Cancer 73(9):2432-2443; Rettig et al. (1988) Proc. Natl. Acad.Sci. USA 85:3110-3114; Campbell et al. (1991) Cancer Res. 51:5329-5338;Coney et al. (1991) Cancer Res. 51:6125-6132; Weitman et al. (1992)Cancer Res. 52:3396-3401; Garin-Chesa et al. (1993) Am. J. Pathol.142:557-567; Holm et al. (1994) APMIS 102:413-419; Franklin et al.(1994) Int. J. Cancer 8 (Suppl.):89-95; Miotti et al. (1987) Int. J.Cancer 39:297-303; and Vegglan et al. (1989) Tumori 75:510-513). FR-α isoverexpressed in greater than 90% of ovarian carcinomas (Sudimack andLee (2000) Adv. Drug Deliv. Rev. 41(2):147-62). FR-α generally attachesto the cell surface membrane via a GPI anchor. GPI anchors containoligosaccharides and inositol phospholipids.

In 1987, Miotti et al. described three new monoclonal antibodies thatrecognized antigens on human ovarian carcinoma cells (Miotti et al.(1987) Int. J. Cancer 39(3):297-303). One of these was designated MOv18,which recognizes a 38 kDa protein on the surface of choriocarcinomacells. MOv18 is a murine, IgG1, kappa antibody and mediates specificcell lysis of the ovarian carcinoma cell line, IGROV1. Alberti et al.((1990) Biochem. Biophys. Res. Commun. 171(3):1051-1055) showed that theantigen recognized by MOv18 was a GPI-linked protein. This wassubsequently identified as the human folate binding protein (Coney etal. (1991) Cancer Res. 51(22):6125-6132). Tomassetti et al. showed thatMOv18 recognizes a soluble form and a GPI-anchored form of the folatebinding protein in IGROV1 cells (Tomassetti et al. (1993) FEBS Lett.317(1-2):143-146). Subsequent work combined the variable regions of themouse MOv18 with human IgG1 (kappa) constant region to create achimerized MOv18 antibody. The chimerized antibody mediated higher andmore specific lysis of IGROV 1 cells at 10-100-fold lower antibodyconcentrations (Coney et al. (1994) Cancer Res. 54(9):2448-2455). The 38kDa antigen appears to be the monomeric form of FR-α.

U.S. Pat. No. 5,952,484 describes a humanized antibody that binds to a38 kDa protein (FR-α). The antibody was named LK26. The original mousemonoclonal antibody was described by Rettig in European PatentApplication No. 86104170.5 (published as EP0197435 and issued in theU.S. as U.S. Pat. No. 4,851,332).

Ovarian cancer is a major cause of death due to gynecologicalmalignancy. Although chemotherapy is the recommended treatment and hasenjoyed some success, the 5-year survival rate is still less than 40%.

A difficult problem in antibody therapy in cancer is that often thetarget of the antibody is expressed by normal tissues as well ascancerous tissues. Thus, the antibodies that are used to kill cancercells also have a deleterious effect on normal cells. Finding uniquetargets or targets that are preferentially expressed in cancer tissueshas proven difficult in many cancers. Identification of preferentiallyexpressed targets and the ability to block the biological activity ofsuch targets may be an effective treatment for cancer. As such, moreeffective antibody therapies for ovarian and other FR-α-bearing cancersthat avoids or minimizes reactivity with normal tissues are needed.

SUMMARY OF THE INVENTION

In some embodiments, the invention provides antibodies that specificallybind to FR-α. The antibodies of the invention preferably block abiological activity of FR-α. In some embodiments, the invention providesantibody-producing cells and compositions of antibodies thatspecifically bind to FR-α wherein the cells and compositions aresubstantially free of FR-α binding competitors. In some embodiments,antibody-producing cells that produce antibodies comprisingsubstantially only antibody of the invention are provided. In preferredembodiments, the antibodies of the invention bind FR-α with a bindingaffinity of at least about 1×10⁻⁷ M, at least about 1×10⁻⁸ M, at leastabout 1×10⁻⁹ M, and most preferably at least about 1×10⁻¹⁰ M.

It has been discovered that tumors that overexpress FR-α tend to favorthe formation of multimeric forms of FR-α, for example tetramers.Without wishing to be bound by any particular theory, it is believedthat the formation of the multimeric form of FR-α is driven by a masseffect due to the accumulation of larger amounts of FR-α on the surfaceof tumor cells. Previously, other researchers only found highermolecular weight species of FR-α in gel filtration assays whichrepresented FR-α inserted into Triton X-100 micelles via theirhydrophobic tails (Holm et al. (1997) Biosci. Reports 17(4):415-427). Insome embodiments, the invention provides antibodies that specificallybind to the multimeric form of FR-α and not the monomeric form.

In some embodiments, the antibodies of the invention (a) bind to anepitope of FR-α other than the epitope bound by antibody LK26; (b) bindFR-α with greater affinity than antibody LK26; (c) out-compete antibodyLK26 for binding to the multimeric form of FR-α and thereby block thebiological activity of FR-α; and/or (d) are purified relative to LK26.

In some embodiments, the antibodies of the invention recognize adisulfide-dependent epitope.

Some embodiments of the invention relate to antibodies comprising aheavy chain comprising an amino acid sequence of SEQ ID NO:5. In someembodiments, the heavy chain comprises an amino acid sequence of SEQ IDNO:6.

In some embodiments, the antibodies of the invention comprise a lightchain comprising the amino acid sequence of SEQ ID NO:2. In someembodiments of the invention, the antibodies comprise a light chaincomprising the amino acid sequence of SEQ ID NO:3.

The invention further provides antibodies comprising a heavy chaincomprising an amino acid of SEQ ID NO:5 or SEQ ID NO:6 and a light chaincomprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3. Theantibodies of the invention preferably comprise a heavy chain comprisingan amino acid sequence of SEQ ID NO:5 and a light chain comprising anamino acid sequence of SEQ ID NO:2 and more preferably comprise a heavychain comprising an amino acid sequence of SEQ ID NO:6 and a light chaincomprising an amino acid sequence of SEQ ID NO:3. In some embodiments ofthe invention, the heavy chain of the antibody is encoded by a nucleicacid comprising the nucleotide sequence of SEQ ID NO:7. In someembodiments of the invention, the light chain of the antibody is encodedby a nucleic acid comprising the nucleotide sequence of SEQ ID NO:8.

The antibodies of the invention may be chimeric antibodies, including,but not limited to human-mouse chimeric antibodies. The antibodies ofthe invention may also be humanized antibodies. The invention alsoprovides: cells, including hybridoma cells, that express the antibodiesof the invention; polynucleotides that encode the antibodies of theinvention; vectors comprising the polynucleotides that encode theantibodies of the invention; and expression cells comprising the vectorsof the invention.

The invention also provides methods of producing an antibody thatspecifically binds to FR-α. In some embodiments, the method comprisesthe step of culturing the antibody-producing cells of the invention. Thecells of the invention may be insect cells or animal cells, preferably,mammalian cells.

The invention further provides methods of inhibiting the growth ofdysplastic cells associated with increased expression of FR-α comprisingadministering to a patient with such dysplastic cells a compositioncomprising an antibody of the invention. The antibody preferably blocksa biological activity of FR-α. The methods may be used for variousdysplastic conditions, such as, but not limited to ovarian, breast,renal, colorectal, lung, endometrial, or brain cancer. In preferredembodiments, the patients are human patients. In some embodiments, theantibodies are conjugated to cytotoxic agents such as, but not limitedto radionuclides, toxins, and chemotherapeutic agents. In someembodiments, the antibodies are co-administered with an antifolateagent. The antifolate agent and antibody of the invention may beadministered at the same time or simultaneously (that is, together), orin any order.

The invention also provides methods for decreasing the growth of cancercells using monoclonal antibodies that specifically bind to FR-α,preferably mammalian FR-α. The methods of the invention may be used tomodulate the growth of cancer cells and the progression of cancer inmammals, including humans. The cancer cells that may be inhibitedinclude all cancer cells that have an increased expression of FR-α inrelation to normal human tissues, such as but not limited to ovarian,breast, renal, colorectal, lung, endometrial, or brain cancer cells.

Also provided by the invention are compositions of antibodies of theinvention. In preferred embodiments, the compositions are substantiallypure. Substantially pure compositions of antibodies of the inventionpreferably comprise at least about 90%, more preferably at least about95%, even more preferably at least about 99%, and most preferably about100% by weight of antibodies of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a western blot of tumor cells showing the tetrameric andmonomeric forms of FR-α.

FIG. 2 shows a western blot of Escherichia coli-expressed FR-α.

FIG. 3 shows a western blot of FR-α solubilized in the presence orabsence of Triton X-100.

FIG. 4 illustrates a screening method for identifying antibody-producingcells of the invention.

FIG. 5A illustrates a sequence alignment of light chain of an anti-FR-αantibody of the invention having an amino acid sequence of SEQ ID NO:3and the light chain of an aberrant translation product having an aminoacid sequence of SEQ ID NO: 24. FIG. 5B illustrates a sequence alignmentof the nucleic acid sequence of a light chain of an anti-FR-α antibodyof the invention having a sequence of SEQ ID NO:8 and a nucleic acidsequence encoding the aberrant translation product having a sequence ofSEQ ID NO:25.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The reference works, patents, patent applications, and scientificliterature, including accession numbers to GenBank database sequencesthat are referred to herein establish the knowledge of those with skillin the art and are hereby incorporated by reference in their entirety tothe same extent as if each was specifically and individually indicatedto be incorporated by reference. Any conflict between any referencecited herein and the specific teachings of this specification shall beresolved in favor of the latter. Likewise, any conflict between anart-understood definition of a word or phrase and a definition of theword or phrase as specifically taught in this specification shall beresolved in favor of the latter.

Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York (1998); Sambrook et al. MOLECULAR CLONING: A LABORATORYMANUAL, 2D ED., Cold Spring Harbor Laboratory Press, Plainview, N.Y.(1989); Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODSIN BIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed.,DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford (1991).

As used herein, the term “epitope” refers to the portion of an antigento which a monoclonal antibody specifically binds.

As used herein, the term “conformational epitope” refers to adiscontinuous epitope formed by a spatial relationship between aminoacids of an antigen other than an unbroken series of amino acids.

As used herein, the term “multimeric” refers to a grouping of two ormore identical or nearly identical units. As used herein, the term“tetrameric” refers to a grouping of four, identical or nearly identicalunits.

As used herein, the term “monomeric” refers to a single unit of a matureprotein that assembles in groups with other units.

As used herein, the term “inhibition of growth of dysplastic cells invitro” means a decrease in the number of tumor cells, in culture, by atleast about 5%, preferably about 10%, more preferably about 20%, morepreferably about 30%, more preferably about 40%, more preferably about50%, more preferably about 60%, more preferably about 70%, morepreferably about 80%, more preferably about 90%, more preferably about95%, more preferably about 99%, and most preferably 100%. In vitroinhibition of tumor cell growth may be measured by assays known in theart, such as the GEO cell soft agar assay.

As used herein, the term “inhibition of growth of dysplastic cells invivo” means a decrease in the number of tumor cells, in an animal, by atleast about 5%, preferably about 10%, more preferably about 20%, morepreferably about 30%, more preferably about 40%, more preferably about50%, more preferably about 60%, more preferably about 70%, morepreferably about 80%, more preferably about 90%, more preferably about95%, more preferably about 99%, and most preferably 100%. In vivomodulation of tumor cell growth may be measured by assays known in theart, for example but not limited to using the Response EvaluationCriteria in Solid Tumors (RECIST) parameters (available online throughthe National Cancer Institute Cancer Therapy Evaluation Program).

As used herein, “dysplastic cells” refer to cells that exhibit abnormalgrowth properties, such as but not limited to growth in soft agar, lackof contact inhibition, failure to undergo cell cycle arrest in theabsence of serum, and formation of tumors when injected intoimmune-compromised mice. Dysplastic cells include, but are not limitedto tumors, hyperplasia, and the like.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops an abnormal condition.

The term “treating” refers to having a therapeutic effect and at leastpartially alleviating or abrogating an abnormal condition in theorganism. Treating includes inhibition of tumor growth, maintenance ofinhibited tumor growth, and induction of remission.

The term “therapeutic effect” refers to the inhibition of an abnormalcondition. A therapeutic effect relieves to some extent one or more ofthe symptoms of the abnormal condition. In reference to the treatment ofabnormal conditions, a therapeutic effect can refer to one or more ofthe following: (a) an increase or decrease in the proliferation, growth,and/or differentiation of cells; (b) inhibition (i.e., slowing orstopping) of growth of tumor cells in vivo (c) promotion of cell death;(d) inhibition of degeneration; (e) relieving to some extent one or moreof the symptoms associated with the abnormal condition; and (f)enhancing the function of a population of cells. The monoclonalantibodies and derivatives thereof described herein effectuate thetherapeutic effect alone or in combination with conjugates or additionalcomponents of the compositions of the invention.

As used herein, the term “inhibits the progression of cancer” refers toan activity of a treatment that slows the modulation of neoplasticdisease toward end-stage cancer in relation to the modulation towardend-stage disease of untreated cancer cells.

As used herein “blocks a biological activity of FR-α” refers to theability of the antibodies (or fragments thereof) of the invention toprevent folate binding to FR-α, to prevent the uptake of folate bycells, or to inhibit signal transduction in the cell triggered byfolate.

As used herein, the term “about” refers to an approximation of a statedvalue within an acceptable range. Preferably the range is +/−5% of thestated value.

As used herein, the term “neoplastic disease” refers to a conditionmarked by abnormal proliferation of cells of a tissue.

As used herein, the term “wild-type” refers to a native sequence, forexample, a native nucleic acid sequence encoding or amino acid sequenceof a heavy or light chain of the antibodies of the invention. Examplesof wild-type sequences of the invention include the sequences of SEQ IDNOs:1-8.

As used herein, the term “FR-α binding competitors” refers to aberranttranscripts of the nucleic acids encoding antibodies of the inventionand aberrant translation products of the antibodies of the inventionthat do not have the biological properties of the anti-FR-α antibodiesof the invention (e.g., antigen binding affinity, ability to block abiological activity of FR-α). For example, an aberrant transcript maycontain a deletion, a frameshift, a nonsense mutation, or a missensemutation. An example of an aberrant translation product is analternative splice variant. An example of a FR-α binding competitor isan antibody comprising a light chain having an amino acid sequence ofSEQ ID NO:24:

MGWSCIILFLVATATGVHSDIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPAASSQRTSPPTTANSGVVTRTC TRSAKGPRWKSNELWLHHLSSSSRHLMSS.

The light chain of such an FR-α binding competitor may be encoded by anucleic acid having a nucleic acid sequence of SEQ ID NO:25:

ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCGACATCCAGCTGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGTGTCAGCTCAAGTATAAGTTCCAACAACTTGCACTGGTACCAGCAGAAGCCCGCAGCCTCCAGCCAGAGGACATCGCCACCTACTACTGCCAACAGTGGAGTAGTTACCCGTACATGTACACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGTTAA.

As used herein, the term “purified” means a condition of beingsufficiently separated from other proteins or nucleic acids with whichit would naturally be associated, so as to exist in “substantially pure”form. “Purified” is not meant to exclude artificial or syntheticmixtures with other compounds or materials, or the presence ofimpurities that do not interfere with the fundamental activity, and thatmay be present, for example, due to incomplete purification, addition ofstabilizers, or compounding into, for example, immunogenic preparationsor pharmaceutically acceptable preparations. A “purified” antibodypreferably means an antibody substantially free of FR-α bindingcompetitors. The term “substantially pure” means comprising at leastabout 50-60% by weight of a given material (e.g., nucleic acid, protein,etc.). More preferably, the preparation comprises at least about 75% byweight, and most preferably about 90-95% by weight of the givencompound. Purity is measured by methods appropriate for the givenmaterial (e.g., chromatographic methods, agarose or polyacrylamide gelelectrophoresis, HPLC analysis, and the like).

As used herein, the phrase “substantially free of FR-α bindingcompetitors” refers to a condition of having less than about 50%, morepreferably less than about 40%, more preferably less than about 30%,more preferably less than about 20%, more preferably less than about10%, more preferably less than about 5%, more preferably less than about1%, more preferably less than about 0.5%, and most preferably about 0%by weight of FR-α binding competitors.

Antibodies

The antibodies of the invention specifically bind folate receptor-alpha(FR-α). In some embodiments, the antibodies of the inventionspecifically bind a monomeric form of FR-α. In some embodiments, theantibodies of the invention specifically bind a multimeric form of FR-α(e.g., a tetrameric form) and not the monomeric form of FR-α. Preferredantibodies of the invention block a biological activity of FR-α. Inpreferred embodiments, the antibodies block a biological activity ofFR-α on FR-α-bearing cells. Antibodies of the invention preferablyinduce antibody-dependent cellular cytotoxicity (ADCC) of FR-α-bearingcells. Examples of FR-α-bearing cells include but are not limited toovarian, lung, breast, brain, renal, colorectal, and endometrial cancercells.

Preferred antibodies, and antibodies suitable for use in the method ofthe invention, include, for example, fully human antibodies, humanantibody homologs, humanized antibody homologs, chimeric antibodyhomologs, Fab, Fab′, F(ab′)₂ and F(v) antibody fragments, single chainantibodies, and monomers or dimers of antibody heavy or light chains ormixtures thereof. Antibodies of the invention are preferably monoclonalantibodies.

The antibodies of the invention may include intact immunoglobulins ofany isotype including types IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof). The antibodies preferably include intact IgG and morepreferably IgG1. The light chains of the immunoglobulin may be kappa orlambda. The light chains are preferably kappa.

The antibodies of the invention include portions of intact antibodiesthat retain antigen-binding specificity, for example, Fab fragments,Fab′ fragments, F(ab′)₂ fragments, F(v) fragments, heavy chain monomersor dimers, light chain monomers or dimers, dimers consisting of oneheavy and one light chain, and the like. Thus, antigen bindingfragments, as well as full-length dimeric or trimeric polypeptidesderived from the above-described antibodies are themselves useful.

A “chimeric antibody” is an antibody produced by recombinant DNAtechnology in which all or part of the hinge and constant regions of animmunoglobulin light chain, heavy chain, or both, have been substitutedfor the corresponding regions from another animal's immunoglobulin lightchain or heavy chain. In this way, the antigen-binding portion of theparent monoclonal antibody is grafted onto the backbone of anotherspecies' antibody. One approach, described in EP 0239400 to Winter etal. describes the substitution of one species' complementaritydetermining regions (CDRs) for those of another species, such assubstituting the CDRs from human heavy and light chain immunoglobulinvariable region domains with CDRs from mouse variable region domains.These altered antibodies may subsequently be combined with humanimmunoglobulin constant regions to form antibodies that are human exceptfor the substituted murine CDRs which are specific for the antigen.Methods for grafting CDR regions of antibodies may be found, for examplein Riechmann et al. (1988) Nature 332:323-327 and Verhoeyen et al.(1988) Science 239:1534-1536.

The direct use of rodent monoclonal antibodies (MAbs) as humantherapeutic agents led to human anti-rodent antibody (“HARA”) (forexample, human anti-mouse antibody (“HAMA”)) responses which occurred ina significant number of patients treated with the rodent-derivedantibody (Khazaeli, et al., (1994) Immunother. 15:42-52). Chimericantibodies containing fewer murine amino acid sequences are believed tocircumvent the problem of eliciting an immune response in humans.

Refinement of antibodies to avoid the problem of HARA responses led tothe development of “humanized antibodies.” Humanized antibodies areproduced by recombinant DNA technology, in which at least one of theamino acids of a human immunoglobulin light or heavy chain that is notrequired for antigen binding has been substituted for the correspondingamino acid from a nonhuman mammalian immunoglobulin light or heavychain. For example, if the immunoglobulin is a mouse monoclonalantibody, at least one amino acid that is not required for antigenbinding is substituted using the amino acid that is present on acorresponding human antibody in that position. Without wishing to bebound by any particular theory of operation, it is believed that the“humanization” of the monoclonal antibody inhibits human immunologicalreactivity against the foreign immunoglobulin molecule.

As a non-limiting example, a method of performing complementaritydetermining region (CDR) grafting may be performed by sequencing themouse heavy and light chains of the antibody of interest that binds tothe target antigen (e.g., FR-α) and genetically engineering the CDR DNAsequences and imposing these amino acid sequences to corresponding humanV regions by site directed mutagenesis. Human constant region genesegments of the desired isotype are added, and the “humanized” heavy andlight chain genes are co-expressed in mammalian cells to produce solublehumanized antibody. A typical expression cell is a Chinese Hamster Ovary(CHO) cell. Suitable methods for creating the chimeric antibodies may befound, for example, in Jones et al. (1986) Nature 321:522-525; Riechmann(1988) Nature 332:323-327; Queen et al. (1989) Proc. Nat. Acad. Sci. USA86:10029; and Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833.

Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029-10033 and WO90/07861 describe the preparation of a humanized antibody. Human andmouse variable framework regions were chosen for optimal proteinsequence homology. The tertiary structure of the murine variable regionwas computer-modeled and superimposed on the homologous human frameworkto show optimal interaction of amino acid residues with the mouse CDRs.This led to the development of antibodies with improved binding affinityfor antigen (which is typically decreased upon making CDR-graftedchimeric antibodies). Alternative approaches to making humanizedantibodies are known in the art and are described, for example, inTempest (1991) Biotechnology 9:266-271.

“Single chain antibodies” refer to antibodies formed by recombinant DNAtechniques in which immunoglobulin heavy and light chain fragments arelinked to the Fv region via an engineered span of amino acids. Variousmethods of generating single chain antibodies are known, including thosedescribed in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward etal. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

The antibodies of the invention may be used alone or as immunoconjugateswith a cytotoxic agent. In some embodiments, the agent is achemotherapeutic agent. In some embodiments, the agent is aradioisotope, including, but not limited to Lead-212, Bismuth-212,Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188,Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-111, andfissionable nuclides such as Boron-10 or an Actinide. In otherembodiments, the agent is a toxin or cytotoxic drug, including but notlimited to ricin, modified Pseudomonas enterotoxin A, calicheamicin,adriamycin, 5-fluorouracil, and the like. Methods of conjugation ofantibodies and antibody fragments to such agents are known in theliterature.

The antibodies of the invention include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody from bindingto its epitope. Examples of suitable derivatives include, but are notlimited to fucosylated antibodies and fragments, glycosylated antibodiesand fragments, acetylated antibodies and fragments, pegylated antibodiesand fragments, phosphorylated antibodies and fragments, and amidatedantibodies and fragments. The antibodies and derivatives thereof of theinvention may themselves by derivatized by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherproteins, and the like. In some embodiments of the invention, at leastone heavy chain of the antibody is fucosylated. In some embodiments, thefucosylation is N-linked. In some preferred embodiments, at least oneheavy chain of the antibody comprises a fucosylated, N-linkedoligosaccharide.

The antibodies of the invention include variants having single ormultiple amino acid substitutions, deletions, additions, or replacementsthat retain the biological properties (e.g., block a biological activityof FR-α, binding affinity) of the antibodies of the invention. Theskilled person can produce variants having single or multiple amino acidsubstitutions, deletions, additions or replacements. These variants mayinclude, inter alia: (a) variants in which one or more amino acidresidues are substituted with conservative or nonconservative aminoacids, (b) variants in which one or more amino acids are added to ordeleted from the polypeptide, (c) variants in which one or more aminoacids include a substituent group, and (d) variants in which thepolypeptide is fused with another peptide or polypeptide such as afusion partner, a protein tag or other chemical moiety, that may conferuseful properties to the polypeptide, such as, for example, an epitopefor an antibody, a polyhistidine sequence, a biotin moiety and the like.Antibodies of the invention may include variants in which amino acidresidues from one species are substituted for the corresponding residuein another species, either at the conserved or nonconserved positions.In another embodiment, amino acid residues at nonconserved positions aresubstituted with conservative or nonconservative residues. Thetechniques for obtaining these variants, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to the person having ordinary skill in the art.Antibodies of the invention also include antibody fragments. A“fragment” refers to polypeptide sequences which are preferably at leastabout 40, more preferably at least to about 50, more preferably at leastabout 60, more preferably at least about 70, more preferably at leastabout 80, more preferably at least about 90, and more preferably atleast about 100 amino acids in length, and which retain some biologicalactivity or immunological activity of the full-length sequence, forexample, the ability to block a biological activity of FR-α and/or FR-αbinding affinity.

The invention also encompasses fully human antibodies such as thosederived from peripheral blood mononuclear cells of ovarian, breast,renal, colorectal, lung, endometrial, or brain cancer patients. Suchcells may be fused with myeloma cells, for example, to form hybridomacells producing fully human antibodies against FR-α.

In preferred embodiments of the invention, the antibody comprises alight chain comprising an amino acid sequence of SEQ ID NO:1:

DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQ QWSSYPYMYTFGQGTKVEIK.In some preferred embodiments, the antibody of the invention comprises alight chain comprising an amino acid sequence of SEQ ID NO:2:

DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.In some preferred embodiments, the antibody of the invention comprises alight chain comprising an amino acid sequence of SEQ ID NO:3:

MGWSCIILFLVATATGVHSDIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

(leader sequence underlined).

Also within the scope of the invention are antibodies comprising a heavychain comprising an amino acid sequence of SEQ ID NO:4:

EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSS.In some preferred embodiments of the invention, the antibodies of theinvention comprise a heavy chain comprising an amino acid sequence ofSEQ ID NO:5:

EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.In some preferred embodiments of the invention, the heavy chain of theantibody comprises an amino acid sequence of SEQ ID NO:6:

MGWSCIILFLVATATGVHSEVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(leader sequence underlined).

In some embodiments of the invention, the antibody comprises a heavychain comprising an amino acid sequence of SEQ ID NO:4, 5, or 6 and alight chain comprising an amino acid sequence of SEQ ID NO:1, 2, or 3.In more preferred embodiments, the antibody comprises a heavy chaincomprising an amino acid sequence of SEQ ID NO:5 and a light chaincomprising an amino acid sequence of SEQ ID NO:2. In some embodiments ofthe invention, the antibody comprises a heavy chain comprising an aminoacid sequence SEQ ID NO:6 and a light chain comprising an amino acidsequence of SEQ ID NO:3.

The antibodies of the invention are preferably nontoxic as demonstrated,for example, in in vivo toxicology studies.

The antibodies and derivatives thereof of the invention have bindingaffinities that include a dissociation constant (K_(d)) of less than1×10⁻². In some embodiments, the K_(d) is less than 1×10⁻³. In otherembodiments, the K_(d) is less than 1×10⁻⁴. In some embodiments, theK_(d) is less than 1×10⁻⁵. In still other embodiments, the K_(d) is lessthan 1×10⁻⁶. In other embodiments, the K_(d) is less than 1×10⁻⁷. Inother embodiments, the K_(d) is less than 1×10⁻⁸. In other embodiments,the K_(d) is less than 1×10⁻⁹. In other embodiments, the K_(d) is lessthan 1×10⁻¹⁰. In still other embodiments, the K_(d) is less than1×10⁻¹¹. In some embodiments, the K_(d) is less than 1×10⁻¹². In otherembodiments, the K_(d) is less than 1×10⁻¹³. In other embodiments, theK_(d) is less than 1×10⁻¹⁴. In still other embodiments, the K_(d) isless than 1×10⁻¹⁵.

Without wishing to be bound by any particular theory, it is believedthat the antibodies of some embodiments of the invention areparticularly useful in binding the multimeric form of FR-α due to anincreased avidity of the antibody as both “arms” of the antibody (Fabfragments) bind to separate FR-α molecules that make up the multimer.This leads to a decrease in the dissociation (K_(d)) of the antibody andan overall increase in the observed affinity (K_(D)).

Nucleic Acids

The invention also includes nucleic acids encoding the heavy chainand/or light chain of the anti-FR-α antibodies of the invention.“Nucleic acid” or a “nucleic acid molecule” as used herein refers to anyDNA or RNA molecule, either single- or double-stranded and, ifsingle-stranded, the molecule of its complementary sequence in eitherlinear or circular form. In discussing nucleic acid molecules, asequence or structure of a particular nucleic acid molecule may bedescribed herein according to the normal convention of providing thesequence in the 5′ to 3′ direction. In some embodiments of theinvention, nucleic acids are “isolated.” This term, when applied to DNA,refers to a DNA molecule that is separated from sequences with which itis immediately contiguous in the naturally occurring genome of theorganism in which it originated. For example, an “isolated nucleic acid”may comprise a DNA molecule inserted into a vector, such as a plasmid orvirus vector, or integrated into the genomic DNA of a prokaryotic oreukaryotic cell or host organism. When applied to RNA, the term“isolated nucleic acid” refers primarily to an RNA molecule encoded byan isolated DNA molecule as defined above. Alternatively, the term mayrefer to an RNA molecule that has been sufficiently separated from othernucleic acids with which it would be associated in its natural state(i.e., in cells or tissues). An isolated nucleic acid (either DNA orRNA) may further represent a molecule produced directly by biological orsynthetic means and separated from other components present during itsproduction.

Nucleic acids of the invention include nucleic acids having at least80%, more preferably at least about 90%, more preferably at least about95%, and most preferably at least about 98% homology to nucleic acids ofthe invention. The terms “percent similarity”, “percent identity” and“percent homology” when referring to a particular sequence are used asset forth in the University of Wisconsin GCG software program. Nucleicacids of the invention also include complementary nucleic acids. In someinstances, the sequences will be fully complementary (no mismatches)when aligned. In other instances, there may be up to about a 20%mismatch in the sequences.

Nucleic acids of the invention also include fragments of the nucleicacids of the invention. A “fragment” refers to a nucleic acid sequencethat is preferably at least about 10 nucleic acids in length, morepreferably about 40 nucleic acids, and most preferably about 100 nucleicacids in length. A “fragment” can also mean a stretch of at least about100 consecutive nucleotides that contains one or more deletions,insertions, or substitutions. A “fragment” can also mean the wholecoding sequence of a gene and may include 5′ and 3′ untranslatedregions.

The encoded antibody light chain preferably comprises an amino acidsequence of SEQ ID NO:1, 2, or 3. The encoded antibody heavy chainpreferably comprises an amino acid sequence of SEQ ID NO:4, 5, or 6. Insome embodiments of the invention, the heavy chain of the antibody isencoded by a nucleic acid comprising the nucleotide sequence of SEQ IDNO:7:

ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCGAGGTCCAACTGGTGGAGAGCGGTGGAGGTGTTGTGCAACCTGGCCGGTCCCTGCGCCTGTCCTGCTCCGCATCTGGCTTCACCTTCAGCGGCTATGGGTTGTCTTGGGTGAGACAGGCACCTGGAAAAGGTCTTGAGTGGGTTGCAATGATTAGTAGTGGTGGTAGTTATACCTACTATGCAGACAGTGTGAAGGGTAGATTTGCAATATCGCGAGACAACGCCAAGAACACATTGTTCCTGCAAATGGACAGCCTGAGACCCGAAGACACCGGGGTCTATTTTTGTGCAAGACATGGGGACGATCCCGCCTGGTTCGCTTATTGGGGCCAAGGGACCCCGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAATGA.In some embodiments of the invention, the light chain of the anti-folatereceptor-α antibody is encoded by a nucleic acid sequence of SEQ IDNO:8:

ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCGACATCCAGCTGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGTGTCAGCTCAAGTATAAGTTCCAACAACTTGCACTGGTACCAGCAGAAGCCAGGTAAGGCTCCAAAGCCATGGATCTACGGCACATCCAACCTGGCTTCTGGTGTGCCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTACACCTTCACCATCAGCAGCCTCCAGCCAGAGGACATCGCCACCTACTACTGCCAACAGTGGAGTAGTTACCCGTACATGTACACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGA GCTTCAACAGGGGAGAGTGTTAA.In some embodiments of the invention are provided nucleic acids encodingboth a heavy chain and a light chain of an antibody of the invention.For example, a nucleic acid of the invention may comprise a nucleic acidsequence encoding an amino acid sequence of SEQ ID NO:1, 2, or 3 and anucleic acid sequence encoding an amino acid sequence of SEQ ID NO:4, 5,or 6.

Nucleic acids of the invention can be cloned into a vector. A “vector”is a replicon, such as a plasmid, cosmid, bacmid, phage, artificialchromosome (BAC, YAC) or virus, into which another genetic sequence orelement (either DNA or RNA) may be inserted so as to bring about thereplication of the attached sequence or element. A “replicon” is anygenetic element, for example, a plasmid, cosmid, bacmid, phage,artificial chromosome (BAC, YAC) or virus, that is capable ofreplication largely under its own control. A replicon may be either RNAor DNA and may be single or double stranded. In some embodiments, theexpression vector contains a constitutively active promoter segment(such as but not limited to CMV, SV40, Elongation Factor or LTRsequences) or an inducible promoter sequence such as the steroidinducible pIND vector (Invitrogen), where the expression of the nucleicacid can be regulated. Expression vectors of the invention may furthercomprise regulatory sequences, for example, an internal ribosomal entrysite. The expression vector can be introduced into a cell bytransfection, for example.

Methods of Producing Antibodies to FR-α

The invention also provides methods of producing monoclonal antibodiesthat specifically bind FR-α. Antibodies of the invention may be producedin vivo or in vitro. One strategy for generating antibodies against FR-αinvolves immunizing animals with FR-α. In some embodiments, animals areimmunized with the monomeric or multimeric form of FR-α. Animals soimmunized will produce antibodies against the protein. Standard methodsare known for creating monoclonal antibodies including, but are notlimited to, the hybridoma technique (see Kohler & Milstein, (1975)Nature 256:495-497); the trioma technique; the human B-cell hybridomatechnique (see Kozbor et al. (1983) Immunol. Today 4:72) and the EBVhybridoma technique to produce human monoclonal antibodies (see Cole, etal. in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc.,1985, pp. 77-96).

FR-α may be purified from cells or from recombinant systems using avariety of well-known techniques for isolating and purifying proteins.For example, but not by way of limitation, FR-α may be isolated based onthe apparent molecular weight of the protein by running the protein onan SDS-PAGE gel and blotting the proteins onto a membrane. Thereafter,the appropriate size band corresponding to FR-α may be cut from themembrane and used as an immunogen in animals directly, or by firstextracting or eluting the protein from the membrane. As an alternativeexample, the protein may be isolated by size-exclusion chromatographyalone or in combination with other means of isolation and purification.

The invention also provides methods of producing monoclonal antibodiesthat specifically bind to the multimeric form of FR-α. Multimeric, forexample tetrameric, FR-α may be purified from cells or from recombinantsystems using a variety of well-known techniques for isolating andpurifying proteins. For example, but not by way of limitation,multimeric FR-α may be isolated based on the apparent molecular weightof the protein by running the protein on an SDS-PAGE gel and blottingthe proteins onto a membrane. Thereafter, the appropriate size bandcorresponding to the multimeric form of FR-α may be cut from themembrane and used as an immunogen in animals directly, or by firstextracting or eluting the protein from the membrane. As an alternativeexample, the protein may be isolated by size-exclusion chromatographyalone or in combination with other means of isolation and purification.

Other means of purification are available in such standard referencetexts as Zola, MONOCLONAL ANTIBODIES: PREPARATION AND USE OF MONOCLONALANTIBODIES AND ENGINEERED ANTIBODY DERIVATIVES (BASICS: FROM BACKGROUNDTO BENCH) Springer-Verlag Ltd., New York, 2000; BASIC METHODS INANTIBODY PRODUCTION AND CHARACTERIZATION, Chapter 11, “AntibodyPurification Methods,” Howard and Bethell, Eds., CRC Press, 2000;ANTIBODY ENGINEERING (SPRINGER LAB MANUAL.), Kontermann and Dubel, Eds.,Springer-Verlag, 2001.

For in vivo antibody production, animals are generally immunized withFR-α or an immunogenic portion of FR-α. The antigen is generallycombined with an adjuvant to promote immunogenicity. Adjuvants varyaccording to the species used for immunization. Examples of adjuvantsinclude, but are not limited to: Freund's complete adjuvant (“FCA”),Freund's incomplete adjuvant (“FIA”), mineral gels (e.g., aluminumhydroxide), surface active substances (e.g., lysolecithin, pluronicpolyols, polyanions), peptides, oil emulsions, keyhole limpet hemocyanin(“KLH”), dinitrophenol (“DNP”), and potentially useful human adjuvantssuch as Bacille Calmette-Guerin (“BCG”) and corynebacterium parvum. Suchadjuvants are also well known in the art.

Immunization may be accomplished using well-known procedures. The doseand immunization regimen will depend on the species of mammal immunized,its immune status, body weight, and/or calculated surface area, etc.Typically, blood serum is sampled from the immunized mammals and assayedfor anti-FR-α antibodies using appropriate screening assays as describedbelow, for example.

A common method for producing humanized antibodies is to graft CDRsequences from a MAb (produced by immunizing a rodent host) onto a humanIg backbone, and transfection of the chimeric genes into Chinese HamsterOvary (CHO) cells which in turn produce a functional Ab that is secretedby the CHO cells (Shields, R. L., et al. (1995) Anti-IgE monoclonalantibodies that inhibit allergen-specific histamine release. Int Arch.Allergy Immunol. 107:412-413). The methods described within thisapplication are also useful for generating genetic alterations within Iggenes or chimeric Igs transfected within host cells such as rodent celllines, plants, yeast and prokaryotes (Frigerio L, et al. (2000)Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin inplants. Plant Physiol. 123:1483-1494).

Splenocytes from immunized animals may be immortalized by fusing thesplenocytes (containing the antibody-producing B cells) with an immortalcell line such as a myeloma line. Typically, myeloma cell line is fromthe same species as the splenocyte donor. In one embodiment, theimmortal cell line is sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). In someembodiments, the myeloma cells are negative for Epstein-Barr virus (EBV)infection. In preferred embodiments, the myeloma cells areHAT-sensitive, EBV negative and Ig expression negative. Any suitablemyeloma may be used. Murine hybridomas may be generated using mousemyeloma cell lines (e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines). These murine myeloma lines are available fromthe ATCC. These myeloma cells are fused to the donor splenocytespolyethylene glycol (“PEG”), preferably 1500 molecular weightpolyethylene glycol (“PEG 1500”). Hybridoma cells resulting from thefusion are selected in HAT medium which kills unfused and unproductivelyfused myeloma cells. Unfused splenocytes die over a short period of timein culture. In some embodiments, the myeloma cells do not expressimmunoglobulin genes.

Hybridomas producing a desired antibody which are detected by screeningassays such as those described below may be used to produce antibodiesin culture or in animals. For example, the hybridoma cells may becultured in a nutrient medium under conditions and for a time sufficientto allow the hybridoma cells to secrete the monoclonal antibodies intothe culture medium. These techniques and culture media are well known bythose skilled in the art. Alternatively, the hybridoma cells may beinjected into the peritoneum of an unimmunized animal. The cellsproliferate in the peritoneal cavity and secrete the antibody, whichaccumulates as ascites fluid. The ascites fluid may be withdrawn fromthe peritoneal cavity with a syringe as a rich source of the monoclonalantibody.

Another non-limiting method for producing human antibodies is describedin U.S. Pat. No. 5,789,650 which describes transgenic mammals thatproduce antibodies of another species (e.g., humans) with their ownendogenous immunoglobulin genes being inactivated. The genes for theheterologous antibodies are encoded by human immunoglobulin genes. Thetransgenes containing the unrearranged immunoglobulin encoding regionsare introduced into a non-human animal. The resulting transgenic animalsare capable of functionally rearranging the transgenic immunoglobulinsequences and producing a repertoire of antibodies of various isotypesencoded by human immunoglobulin genes. The B-cells from the transgenicanimals are subsequently immortalized by any of a variety of methods,including fusion with an immortalizing cell line (e.g., a myeloma cell).

Antibodies against FR-α may also be prepared in vitro using a variety oftechniques known in the art. For example, but not by way of limitation,fully human monoclonal antibodies against FR-α may be prepared by usingin vitro-primed human splenocytes (Boerner et al. (1991) J. Immunol.147:86-95).

Alternatively, for example, the antibodies of the invention may beprepared by “repertoire cloning” (Persson et al. (1991) Proc. Nat. Acad.Sci. USA 88:2432-2436; and Huang and Stollar (1991) J. Immunol. Methods141:227-236). Further, U.S. Pat. No. 5,798,230 describes preparation ofhuman monoclonal antibodies from human B antibody-producing B cells thatare immortalized by infection with an Epstein-Barr virus that expressesEpstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2, required forimmortalization, is then inactivated resulting in increased antibodytiters.

In another embodiment, antibodies against FR-α are formed by in vitroimmunization of peripheral blood mononuclear cells (“PBMCs”). This maybe accomplished by any means known in the art, such as, for example,using methods described in the literature (Zafiropoulos et al. (1997) J.Immunological Methods 200:181-190).

Methods for producing antibody-producing cells of the invention alsoinclude methods for developing hypermutable antibody-producing cells bytaking advantage of the conserved mismatch repair (MMR) process of hostcells. Dominant negative alleles of such genes, when introduced intocells or transgenic animals, increase the rate of spontaneous mutationsby reducing the effectiveness of DNA repair and thereby render the cellsor animals hypermutable. Blocking MMR in antibody-producing cells suchas but not limited to: hybridomas; mammalian cells transfected withgenes encoding for Ig light and heavy chains; mammalian cellstransfected with genes encoding for single chain antibodies; eukaryoticcells transfected with Ig genes, can enhance the rate of mutation withinthese cells leading to clones that have enhanced antibody production,cells containing genetically altered antibodies with enhancedbiochemical properties such as increased antigen binding, cells thatproduce antibodies comprising substantially only the antibody of theinvention, and/or cells that are substantially free of FR-α bindingcompetitors. The process of MMR, also called mismatch proofreading, iscarried out by protein complexes in cells ranging from bacteria tomammalian cells. A MMR gene is a gene that encodes for one of theproteins of such a mismatch repair complex. Although not wanting to bebound by any particular theory of mechanism of action, a MMR complex isbelieved to detect distortions of the DNA helix resulting fromnon-complementary pairing of nucleotide bases. The non-complementarybase on the newer DNA strand is excised, and the excised base isreplaced with the appropriate base, which is complementary to the olderDNA strand. In this way, cells eliminate many mutations that occur as aresult of mistakes in DNA replication.

Dominant negative alleles cause a MMR defective phenotype even in thepresence of a wild-type allele in the same cell. An example of adominant negative allele of a MMR gene is the human gene hPMS2-134,which carries a truncating mutation at codon 134. The mutation causesthe product of this gene to abnormally terminate at the position of the134th amino acid, resulting in a shortened polypeptide containing theN-terminal 133 amino acids. Such a mutation causes an increase in therate of mutations, which accumulate in cells after DNA replication.Expression of a dominant negative allele of a mismatch repair generesults in impairment of mismatch repair activity, even in the presenceof the wild-type allele. Any allele which produces such effect can beused in this invention. Dominant negative alleles of a MMR gene can beobtained from the cells of humans, animals, yeast, bacteria, or otherorganisms. Such alleles can be identified by screening cells fordefective MMR activity. Cells from animals or humans with cancer can bescreened for defective mismatch repair. Cells from colon cancer patientsmay be particularly useful. Genomic DNA, cDNA, or mRNA from any cellencoding a MMR protein can be analyzed for variations from the wild typesequence. Dominant negative alleles of a MMR gene can also be createdartificially, for example, by producing variants of the hPMS2-134 alleleor other MMR genes. Various techniques of site-directed mutagenesis canbe used. The suitability of such alleles, whether natural or artificial,for use in generating hypermutable cells or animals can be evaluated bytesting the mismatch repair activity caused by the allele in thepresence of one or more wild-type alleles, to determine if it is adominant negative allele. Examples of mismatch repair proteins andnucleic acid sequences include mouse PMS2 (SEQ ID NOs:9 and 10), humanPMS2 (SEQ ID NOs:11 and 12), human PMS1 (SEQ ID NOs:13 and 14), humanMSH2 (SEQ ID NOs: 15 and 16), human MLH1 (SEQ ID NOs:17 and 18), andhuman PMS2-134 (SEQ ID NOs:19 and 20).

A cell into which a dominant negative allele of a mismatch repair genehas been introduced will become hypermutable. This means that thespontaneous mutation rate of such cells or animals is elevated comparedto cells or animals without such alleles. The degree of elevation of thespontaneous mutation rate can be at least 2-fold, 5-fold, 10-fold,20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold that of thenormal cell or animal. The use of chemical mutagens such as but limitedto methane sulfonate, dimethyl sulfonate, 06-methyl benzadine, MNU, ENU,etc. can be used in MMR defective cells to increase the rates anadditional 10 to 100 fold that of the MMR deficiency itself.

According to one aspect of the invention, a polynucleotide encoding adominant negative form of a MMR protein is introduced into a cell.Preferably the cell produces anti-FR-α antibodies. In some embodiments,the cells produce an antibody comprising a heavy chain comprising anamino acid sequence of SEQ ID NO:4, 5, or 6 and a light chain comprisingan amino acid sequence of SEQ ID NO:1, 2, or 3. In some preferredembodiments, the cells comprise a nucleic acid comprising a nucleotidesequence of SEQ ID NO:7 and/or a nucleotide sequence of SEQ ID NO:8. Thedominant negative MMR gene can be any dominant negative allele encodinga protein which is part of a MMR complex, for example, PMS2, PMS1, MLH1,or MSH2. The dominant negative allele can be naturally occurring or madein the laboratory. The polynucleotide can be in the form of genomic DNA,cDNA, RNA, or a chemically synthesized polynucleotide.

The polynucleotide can be cloned into an expression vector containing aconstitutively active promoter segment (such as but not limited to CMV,SV40, Elongation Factor or LTR sequences) or an inducible promotersequence such as the steroid inducible pIND vector (Invitrogen), wherethe expression of the dominant negative MMR gene can be regulated. Thepolynucleotide can be introduced into the cell by transfection.

According to another aspect of the invention, an immunoglobulin (Ig)gene, a set of Ig genes or a chimeric gene containing whole or parts ofan Ig gene can be transfected into MMR-deficient cell hosts, the cell isgrown and screened for clones with new phenotypes and/or genotypes.MMR-defective cells may be of human, primates, mammals, rodent, plant,yeast or of the prokaryotic kingdom. The gene encoding the Ig of thecell with the new phenotype or genotype may be isolated from therespective clone and introduced into genetically stable cells (i.e.,cells with normal MMR) to provide clones that consistently produce theIg. The method of isolating the Ig gene may be any method known in theart. Introduction of the isolated polynucleotide encoding the Ig mayalso be performed using any method known in the art, including, but notlimited to transfection of an expression vector containing thepolynucleotide encoding the Ig. As an alternative to transfecting an Iggene, a set of Ig genes or a chimeric gene containing whole or parts ofan Ig gene into an MMR-deficient host cell, such Ig genes may betransfected simultaneously with a gene encoding a dominant negativemismatch repair gene into a genetically stable cell to render the cellhypermutable.

Transfection is any process whereby a polynucleotide is introduced intoa cell. The process of transfection can be carried out in a livinganimal, e.g., using a vector for gene therapy, or it can be carried outin vitro, e.g., using a suspension of one or more isolated cells inculture. The cell can be any type of eukaryotic cell, including, forexample, cells isolated from humans or other primates, mammals or othervertebrates, invertebrates, and single celled organisms such asprotozoa, yeast, or bacteria.

In general, transfection will be carried out using a suspension ofcells, or a single cell, but other methods can also be applied as longas a sufficient fraction of the treated cells or tissue incorporates thepolynucleotide so as to allow transfected cells to be grown andutilized. The protein product of the polynucleotide may be transientlyor stably expressed in the cell. Techniques for transfection are wellknown. Available techniques for introducing polynucleotides include butare not limited to electroporation, transduction, cell fusion, the useof calcium chloride, and packaging of the polynucleotide together withlipid for fusion with the cells of interest. Once a cell has beentransfected with the MMR gene, the cell can be grown and reproduced inculture. If the transfection is stable, such that the gene is expressedat a consistent level for many cell generations, then a cell lineresults.

Upon identification of the desired phenotype or trait the organism canthen be genetically stabilized. Cells expressing the dominant negativealleles can be “cured” in that the dominant negative allele can beturned off, if inducible, eliminated from the cell, and the like suchthat the cells become genetically stable and no longer accumulatemutations at the abnormally high rate.

Cells that produce substantially only anti-FR-α antibodies of theinvention or cells that are substantially free of FR-α bindingcompetitors are selected for cloning and expansion according to themethods for determining antibody specificity described herein. Anexample of such a method is illustrated in FIG. 4.

Nucleic acids encoding antibodies of the invention may be recombinantlyexpressed. The expression cells of the invention include any insectexpression cell line known, such as for example, Spodoptera frugiperdacells. The expression cell lines may also be yeast cell lines, such as,for example, Saccharomyces cerevisiae and Schizosaccharomyces pombecells. The expression cells may also be mammalian cells such as, forexample, hybridoma cells (e.g., NS0 cells), Chinese hamster ovary cells,baby hamster kidney cells, human embryonic kidney line 293, normal dogkidney cell lines, normal cat kidney cell lines, monkey kidney cells,African green monkey kidney cells, COS cells, and non-tumorigenic mousemyoblast G8 cells, fibroblast cell lines, myeloma cell lines, mouseNIH/3T3 cells, LMTK31 cells, mouse sertoli cells, human cervicalcarcinoma cells, buffalo rat liver cells, human lung cells, human livercells, mouse mammary tumor cells, TRI cells, MRC 5 cells, and FS4 cells.Nucleic acids of the invention may be introduced into cell bytransfection, for example. Recombinantly expressed antibodies may berecovered from the growth medium of the cells, for example.

In one embodiment of the invention, the procedure for in vitroimmunization is supplemented with directed evolution of the hybridomacells in which a dominant negative allele of a mismatch repair gene suchas PMS1, PMS2, PMS2-134, PMSR2, PMSR3, MLH1, MLH2, MLH3, MLH4, MLH5,MLH6, PMSL9, MSH1, and MSH2 is introduced into the hybridoma cells afterfusion of the splenocytes, or to the myeloma cells before fusion. Cellscontaining the dominant negative mutant will become hypermutable andaccumulate mutations at a higher rate than untransfected control cells.A pool of the mutating cells may be screened, for example, for clonesthat are substantially free of FR-α binding competitors, clones thatproduce higher affinity antibodies, clones that produce higher titers ofantibodies, or clones that simply grow faster or better under certainconditions. The technique for generating hypermutable cells usingdominant negative alleles of mismatch repair genes is described, forexample, in U.S. Pat. No. 6,808,894. Alternatively, mismatch repair maybe inhibited using the chemical inhibitors of mismatch repair describedby Nicolaides et al. in WO 02/054856 “Chemical Inhibitors of MismatchRepair” published Jul. 18, 2002. The technique for enhancing antibodiesusing the dominant negative alleles of mismatch repair genes or chemicalinhibitors of mismatch repair may be applied to mammalian expressioncells expressing cloned immunoglobulin genes as well. Cells expressingthe dominant negative alleles can be “cured” in that the dominantnegative allele can be turned off if inducible, inactivated, eliminatedfrom the cell, and the like, such that the cells become geneticallystable once more and no longer accumulate mutations at the abnormallyhigh rate.

Screening for Antibody Specificity

Screening for antibodies that specifically bind to FR-α may beaccomplished using an enzyme-linked immunosorbent assay (ELISA) in whichmicrotiter plates are coated with FR-α. In some embodiments, antibodiesthat bind FR-α from positively reacting clones can be further screenedfor reactivity in an ELISA-based assay to other folate receptorisoforms, for example, FR-β and/or FR-γ, using microtiter plates coatedwith the other folate receptor isoform(s). Clones that produceantibodies that are reactive to another isoform of folate receptor areeliminated, and clones that produce antibodies that are reactive to FR-αonly may be selected for further expansion and development. Confirmationof reactivity of the antibodies to FR-α may be accomplished, forexample, using a Western Blot assay in which protein from ovarian,breast, renal, colorectal, lung, endometrial, or brain cancer cells andpurified FR-α and other folate receptor isoforms are run on an SDS-PAGEgel, and subsequently are blotted onto a membrane. The membrane may thenbe probed with the putative anti-FR-α antibodies. Reactivity with FR-αand not another folate receptor isoform confirms specificity ofreactivity for FR-α.

In some embodiments, the binding affinity of anti-FR-α antibodies isdetermined Antibodies of the invention preferably have a bindingaffinity to FR-α of at least about 1×10⁻⁷ M, more preferably at leastabout 1×10⁻⁸ M, more preferably at least about 1×10⁻⁹ M, and mostpreferably at least about 1×10⁻¹⁰ M. Preferred antibody-producing cellsof the invention produce substantially only antibodies having a bindingaffinity to FR-α of at least about 1×10⁻⁷ M, more preferably at leastabout 1×10⁻⁸ M, more preferably at least about 1×10⁻⁹ M, and mostpreferably at least about 1×10⁻¹⁰ M. Preferred compositions of theinvention comprise substantially only antibodies having a bindingaffinity to FR-α of at least about 1×10⁻⁷ M, more preferably at leastabout 1×10⁻⁸ M, more preferably at least about 1×10⁻⁹ M, and mostpreferably at least about 1×10⁻¹⁰ M.

In some embodiments, antibodies that bind the multimeric form of FR-αfrom positively reacting clones can be further screened for reactivityin an ELISA-based assay to the monomeric form of FR-α using microtiterplates coated with the monomeric form of FR-α. Clones that produceantibodies that are reactive to the monomeric form of FR-α areeliminated, and clones that produce antibodies that are reactive to themultimeric form only may be selected for further expansion anddevelopment. Confirmation of reactivity of the antibodies to themultimeric form of FR-α may be accomplished, for example, using aWestern Blot assay in which protein from ovarian, breast, renal,colorectal, lung, endometrial, or brain cancer cells and purifiedmultimeric and monomeric FR-α are run on an SDS-PAGE gel under reducingand non-reducing conditions, and subsequently are blotted onto amembrane. The membrane may then be probed with the putativeanti-multimeric FR-α antibodies. Reactivity with the appropriately sizedmultimeric form of FR-α under non-reducing conditions and not the 38 kDaform of FR-α (under reducing or non-reducing conditions) confirmsspecificity of reactivity for the multimeric form of FR-α.

The antibodies of the invention preferably induce antibody-dependentcellular cytotoxicity (ADCC) in FR-α bearing cells. ADCC assays areknown in the art. The method of the invention enabled successfulproduction of an optimized, humanized anti-FR-α antibody with acceptableantigen binding activity (low nanomolar dissociation constant) andproduction rates (>10 pg/cell/day). ADCC assays using human ovariancancer cells as target and peripheral blood mononuclear cells (PBMCs) aseffector cells showed that 200 ng/ml of antibody of the inventionproduced in CHO cells mediated the lysis of 32% of target cells whereaslysis mediated by control IgG₁/κ antibody was only 6% (paired Ttest=0.0008).

Anti-FR-α Antibody-Producing Cells

Antibody-producing cells of the invention include any insect expressioncell line known, such as for example, Spodoptera frugiperda cells. Theexpression cell lines may also be yeast cell lines, such as, forexample, Saccharomyces cerevisiae and Schizosaccharomyces pombe cells.The expression cells may also be mammalian cells such as, for example,hybridoma cells (e.g., NS0 cells), Chinese hamster ovary cells, babyhamster kidney cells, human embryonic kidney line 293, normal dog kidneycell lines, normal cat kidney cell lines, monkey kidney cells, Africangreen monkey kidney cells, COS cells, and non-tumorigenic mouse myoblastG8 cells, fibroblast cell lines, myeloma cell lines, mouse NIH/3T3cells, LMTK31 cells, mouse sertoli cells, human cervical carcinomacells, buffalo rat liver cells, human lung cells, human liver cells,mouse mammary tumor cells, TRI cells, MRC 5 cells, and FS4 cells.

In some preferred embodiments, the antibody-producing cells of theinvention produce antibodies that specifically bind to FR-α. The cellspreferably are substantially free of FR-α binding competitors. Inpreferred embodiments, the antibody-producing cells comprise less thanabout 10%, preferably less than about 5%, more preferably less thanabout 1%, more preferably less than about 0.5%, more preferably lessthan about 0.1%, and most preferably 0% by weight FR-α bindingcompetitors. In some preferred embodiments, the antibodies produced bythe antibody-producing cells are substantially free of FR-α bindingcompetitors. In preferred embodiments, antibodies produced by theantibody-producing cells comprise less than about 10%, preferably lessthan about 5%, more preferably less than about 1%, more preferably lessthan about 0.5%, more preferably less than about 0.1%, and mostpreferably 0% by weight FR-α binding competitors. Preferredantibody-producing cells of the invention produce substantially onlyantibodies having a binding affinity to FR-α of at least about 1×10⁻⁷ M,more preferably at least about 1×10⁻⁸M, more preferably at least about1×10⁻⁹ M, and most preferably at least about 1×10⁻¹⁰ M.

Antibody Purification

Methods of antibody purification are known in the art. In someembodiments of the invention, methods for antibody purification includefiltration, affinity column chromatography, cation exchangechromatography, anion exchange chromatography, and concentration. Thefiltration step preferably comprises ultrafiltration, and morepreferably ultrafiltration and diafiltration. Filtration is preferablyperformed at least about 5-50 times, more preferably 10 to 30 times, andmost preferably 14 to 27 times. Affinity column chromatography, may beperformed using, for example, PROSEP Affinity Chromatography (Millipore,Billerica, Massachusetts). In a preferred embodiment, the affinitychromatography step comprises PROSEP-VA column chromatography. Eluatemay be washed in a solvent detergent. Cation exchange chromatography mayinclude, for example, SP-Sepharose Cation Exchange Chromatography. Anionexchange chromatography may include, for example but not limited to,Q-Sepharose Fast Flow Anion Exchange. The anion exchange step ispreferably non-binding, thereby allowing removal of contaminantsincluding DNA and BSA. The antibody product is preferably nanofiltered,for example, using a Pall DV 20 Nanofilter. The antibody product may beconcentrated, for example, using ultrafiltration and diafiltration. Themethod may further comprise a step of size exclusion chromatography toremove aggregates.

Pharmaceutical Compositions of Antibodies

Another aspect of the invention features a pharmaceutical composition ofanti-FR-α antibodies of the invention. The pharmaceutical compositionsmay be used to inhibit or reduce growth of tumor cells in a patient. Thecompositions of antibodies preferably are substantially free of FR-αbinding competitors. In certain embodiments, the pharmaceuticalcomposition is formulated for administration by injection or infusion.

Pharmaceutical compositions of the invention may further comprise achemotherapeutic or cytotoxic agent. In some embodiments, the antibodyis conjugated to the chemotherapeutic or cytotoxic agent. Suitablechemotherapeutic or cytotoxic agents include but are not limited to aradioisotope, including, but not limited to Lead-212, Bismuth-212,Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188,Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-111, andfissionable nuclides such as Boron-10 or an Actinide. In otherembodiments, the agent is a toxin or cytotoxic drug, including but notlimited to ricin, modified Pseudomonas enterotoxin A, calicheamicin,adriamycin, 5-fluorouracil, and the like. Pharmaceutical compositions ofthe invention may comprise an antifolate compound including but notlimited to 5-fluoro-2′-deoxy-uridine-5′-monophosphate (FdUMP),5-fluorouracil, leucovorin, ZD1649, MTA, GW1843U89, ZD9331, AG337, andPT523.

Pharmaceutical compositions of the invention may be formulated with apharmaceutically acceptable carrier or medium. Suitable pharmaceuticallyacceptable carriers include water, PBS, salt solution (such as Ringer'ssolution), alcohols, oils, gelatins, and carbohydrates, such as lactose,amylose, or starch, fatty acid esters, hydroxymethylcellulose, andpolyvinyl pyrolidine. Such preparations can be sterilized, and ifdesired, mixed with auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, and coloring. Pharmaceutical carriers suitable foruse in the present invention are known in the art and are described, forexample, in Pharmaceutical Sciences (17^(th) Ed., Mack Pub. Co., Easton,Pa.).

Kits

According to yet another aspect of the invention, a kit is provided forinhibiting or reducing growth of tumor cells in a patient. Also providedare kits for identifying the presence of dysplastic cells in vitro or invivo.

The kits of the invention comprise antibody or an antibody compositionof the invention and instructions for using the kit in a method forinhibiting or reducing growth of tumor cells in the patient or in amethod for identifying the presence of dysplastic cells, for example, ina biological sample. The kit may comprise at least one chemotherapeuticor cytotoxic reagent. The kit may comprise an antifolate compound. Thekit may comprise at least one diagnostic reagent. An example of adiagnostic reagent is a detectable label, for example but not limited toa radioactive, fluorescent, or chromophoric agent (e.g., ¹¹¹In-DOTA).The detectable label may comprise an enzyme. The kit may compriseinstructions and/or means for administering the antibody or antibodycomposition, for example, by injection.

Methods of Detecting a Dysplastic Cell

The methods of the invention include methods of detecting dysplastic orcancer cells presenting FR-α on the surface, including but not limitedto ovarian, breast, lung, endometrial, renal, colorectal, or braincarcinoma cells. The method may be performed in vitro on a biologicalsample or in vivo. Methods of detecting dysplastic cells according tothe invention comprise contacting anti-FR-α antibody of the inventionwith a biological sample or administering anti-FR-α antibody of theinvention to a patient, wherein the antibody is labeled with adetectable label, for example but not limited to a radioactive,fluorescent, or chromophoric agent (e.g., ¹¹¹In-DOTA), and determiningbinding of the antibody to cells. The detectable label may be an enzyme.

Methods of Reducing the Growth of Tumor Cells

The methods of the invention are suitable for use in humans andnon-human animals identified as having a neoplastic condition associatedwith an increased expression of FR-α. Non-human animals which benefitfrom the invention include pets, exotic (e.g., zoo animals), anddomestic livestock. Preferably the non-human animals are mammals.

The invention is suitable for use in a human or animal patient that isidentified as having a dysplastic disorder that is marked by increasedexpression of FR-α in the neoplasm in relation to normal tissues. Oncesuch a patient is identified as in need of treatment for such acondition, the method of the invention may be applied to effecttreatment of the condition. Tumors that may be treated include, but arenot limited to ovarian, breast, renal, colorectal, lung, endometrial,brain, fallopian tube, or uterine tumors, and certain leukemia cells. Insome embodiments, the tumor is cisplatin-resistant.

The antibodies and derivatives thereof for use in the invention may beadministered orally in any acceptable dosage form such as capsules,tablets, aqueous suspensions, solutions or the like. The antibodies andderivatives thereof may also be administered parenterally including butnot limited to: subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intranasal, topically,intrathecal, intrahepatic, intralesional, and intracranial injection orinfusion techniques. Generally, the antibodies will be intravenously orintraperitoneally, for example, by injection.

The antibodies and derivatives of the invention may be administeredalone or with a pharmaceutically acceptable carrier, includingacceptable adjuvants, vehicles and excipients, for example, phosphatebuffered saline.

The antibodies and derivatives of the invention may also be administeredwith one or more antifolate compounds that are used to treat cancer. Theantifolate compounds include, but are not limited to5-fluoro-2′-deoxy-uridine-5′-monophosphate (FdUMP); 5-fluorouracil(5-FU); L-5-formyltetrahydrofolate (“leucovorin”);N-[5-(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)-amino)-2-thenyl)]-L-glutamicacid (“ZD1649”; also known as “Tomudex”) (Jackman et al. (1991) CancerRes. 51:5579-5586);N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)-ethyl)-benzoyl]-L-glutamicacid (“multi-targeted antifolate” (MTA) also known as “LY231514,”“ALIMTA,” and “Pemetrexed”)(Taylor et al. (1992) J. Med. Chem.35:4450-4454; Shih et al. (1997) Cancer Res. 57:1116-1123);(S)-2-(5)-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)-methyl)-amino)-oxo-2-isoindolinyl)-glutaricacid (“GW1843U89”) (Hanlon and Ferone (1996) Cancer Res. 56:3301-3306);(2S)-2-{O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-yl-methyl)-N-prop-2-ynyl)amino]benzamido}-4-(tetrazol-5-yl)-butyricacid (“ZD9331”) (Jackman et al. (1997) Clin. Cancer Res. 3:911-921);3,4-dihydro-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazoline (“AG337”also known as “Thymitaq”) (Webber et al. (1996) Cancer Chemother.Pharmacol. 37:509-517; Rafi et al. (1998) J. Clin. Oncol. 16:1331-1341),and N^(α)-(4-amino-4-deoxypteroyl)-N^(δ)-(hemiphthaloyl-L-ornithine)(“PT523”) (Rhee et al. (1994) Mol. Pharmacol. 45:783-791; Rowowsky(1999) Curr. Med. Chem. 6:329-352). The antifolate compounds may beadministered before, after, or simultaneously with the anti-FR-αantibodies of the invention. The amounts of antifolate compounds to beadministered may be the dosages currently used, or may be increased ordecreased, as can readily be determined by a physician based onachieving decreased tumor growth or tumor elimination without causingany untoward effects on the patient.

The effective dosage will depend on a variety of factors. It is wellwithin the purview of a skilled physician to adjust the dosage for agiven patient according to various parameters such as body weight, thegoal of treatment, the highest tolerated dose, the specific formulationused, the route of administration and the like. Generally, dosage levelsof between about 5.88 mg/m² and about 294.12 mg/m² (i.e., 10 to 500 mgantibody) per day of the antibody or derivative thereof are suitable. Insome embodiments, the dose will be about 29.41 mg/m² to about 176.47mg/m² (i.e., 50 to 300 mg antibody) per day of the antibody orderivative thereof. In other embodiments, the dose will be about 58.82mg/m² to about 147.06 mg/m² (i.e., 100 to 250 mg antibody) per day. Instill other embodiments, the dose will be about 88.24 mg/m² to about117.65 mg/m² (i.e., 150 to 200 mg antibody) per day. Dosing may be as abolus or an infusion. Dosages may be given once a day or multiple timesin a day. Further, dosages may be given multiple times of a period oftime. In some embodiments, the doses are given every 1-14 days. In someembodiments, the antibodies or derivatives thereof are given as a doseof about 10 to 500 mg i.p. In other embodiments, the antibodies ofderivatives thereof are provided at about 50 to 300 mg i.v. In stillother embodiments, the antibodies or derivatives thereof are providedsuch that a plasma level of at least about 1 ug/ml is maintained.

Effective treatment may be assessed in a variety of ways. In oneembodiment, effective treatment is determined by a slowed progression oftumor growth. In other embodiments, effective treatment is marked byshrinkage of the tumor (i.e., decrease in the size of the tumordetermined, for example, using Response Evaluation Criteria in SolidTumors (RECIST) available online through the National Cancer InstituteCancer Therapy Evaluation Program). In other embodiments, effectivetreatment is marked by inhibition of metastasis of the tumor. In stillother embodiments, effective therapy is measured by increased well-beingof the patient including such signs as weight gain, regained strength,decreased pain, thriving, and subjective indications from the patient ofbetter health.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

EXAMPLES Example 1 Generation of Anti-FR-α Antibody-Producing Cells

Murine antibody LK26 was raised against choriocarcinoma cell lineLu-75(c). LK26 was humanized by CDR grafting, yielding an IgG (IgG1/κsubtype) expressed in NS0 cell lines, according to the method of U.S.Pat. No. 6,124,106. The NS0 cell line was transfected with a hPMS2-134expression plasmid. The MMR gene was cloned into the pEF expressionvector, which contains the elongation factor promoter upstream of thecloning site followed by a mammalian polyadenylation signal. This vectoralso contains the NEOr gene that allows for selection of cells retainingthis plasmid. Briefly, cells were transfected with 1 μg of each vectorusing polyliposomes following the manufacturer's protocol (LifeTechnologies). Cells were then selected in 0.5 mg/ml of G418 for 10 daysand G418 resistant cells were pooled together to analyze for geneexpression. The pEF construct contains an intron that separates the exon1 of the EF gene from exon 2, which is juxtaposed to the 5′ end of thepolylinker cloning site. This allows for a rapid reverse transcriptasepolymerase chain reaction (RT-PCR) screen for cells expressing thespliced products. Cells were isolated and their RNA extracted using thetrizol method as previously described (Nicolaides N. C., Kinzler, K. W.,and Vogelstein, 8. (1995) Analysis of the 5′ region of PMS2 revealsheterogeneous transcripts and a novel overlapping gene. Genomics29:329-334).

Heavy chain RNA was reverse transcribed using a forward primer(5′-GATCGGATCCACCATGGGATGGAGCTGTATCATCC-3′ (SEQ ID NO:21)) and reverseprimer (5′-CTGATCTAGATCATTTCCCGGGAGACAGGGAGAGGCTCTTCTGCGTGTA-3′ (SEQ IDNO:22)). Light chain RNA was reverse transcribed using a forward primerof SEQ ID NO:21 and a reverse primer(5′-CTGATCTAGATTAACACTCTCCCCTGTTGAAGCTCTT-3′ (SEQ ID NO:23)). PCRreactions were carried out with high fidelity HERCULASE DNA polymerase(STRATAGENE, La Jolla, Calif.). PCR products were digested with BamHIand XbaI and cloned into the same restriction sites of the eukaryoticexpression vectors pEF4 (light chain) and pEF6 (heavy chain). VectorpEF4 (INVITROGEN) is a 5.8 kb vector carrying the zeocin resistance genefor selection of stable transfectants in eukaryotic cells. The cDNAinsert is cloned downstream of hEF-intron 1, and its transcription iscontrolled by the human EF1alpha promoter. Downstream of the cDNA insertis the BGH polyadenylation signal allowing for efficient polyadenylationof the transcript. Vector pEF6 (INVITROGEN) is similar to pEF4 butcarries the blasticidin resistance gene instead of the zeocin resistancegene. The sequence of both strands of the cDNA inserts was verified.

The resulting cDNAs coding for the full-length humanized anti-FR-αantibody heavy and light chains were transfected into CHO-K1 (ATCCCCL-61) cells. CHO-K1 cells were transfected with 0.5 micrograms of eachplasmid using FUGENE transfection reagent (Roche) according to themanufacturer's instructions. Cells were maintained in RPMI1640/10% FBS/2mM L-glutamine. Stable cell lines were selected with Zeocin (200micrograms/milliliter) and Blasticidin (5 micrograms/ milliliter).Expression of antibody was verified by anti-human IgG ELISA. Stablytransfected pools of cells were single cell cloned by limited dilutionand high expressor cell lines were selected. High titers were verifiedin secondary and tertiary screens. The cell line was adapted toserum-free medium (CHO-S-SFMII followed by EX-CELL 302). Antibodyproduction was verified by ELISA. The cell line also was adapted toprotein-free CHO media (CD94111; Irvine Scientific) plus 8 mML-glutamine with a soy hydrolysate pulse at day 2. Cells were stored foruse in liquid nitrogen. The cells were stable for at least 13 passagesin the absence of selection media as determined by FACS analysis. Cellsecretion was stable for at least 20 passages as determined by ELISA.Large scale antibody production is possible. For example, antibody wasproduced in a bioreactor on a scale of 15 L, 70 L, and 340 L.

Example 2 Screening Strategy to Identify Antibody-Producing Clones andCharacterization of Anti-FR-α Antibody

An application of the methods presented within this document is the useof MMR-deficient immunoglobulin-producing cells to create a cell that issubstantially free of FR-α binding competitors or a cell that producessubstantially only the target immunoglobulin, for example, a FR-αantibody of the invention, including but not limited to an antibodycomprising a light chain comprising an amino acid sequence of SEQ IDNO:2 or 3 and a heavy chain comprising an amino acid sequence of SEQ IDNO:5 or 6. FIG. 4 outlines the screening procedure to identify clonesthat produce high affinity MAbs. The assay employs the use of a plateEnzyme Linked Immunosorbant Assay (ELISA) to screen for clones thatproduce high-affinity MAbs. 96-well plates containing singleimmunoglobulin-producing cells are grown in growth medium plus 0.5 mg/mlG418 to ensure clones retain the expression vector. Plates are screenedusing an hIgG plate ELISA, whereby a 96 well plate is coated with FR-α.Alternatively, the plate is coated with a specific antibody against theanti-FR-α antibody. As another alternative in cases in which theimmunoglobulin-producing cell is non-human, the plate may be coated withanti-human IgG1 antibody. Plates are washed 3 times in calcium andmagnesium free phosphate buffered saline solution (PBS−/−) and blockedin 100 μls of PBS−/− with 5% dry milk for 1 hour at room temperature.Wells are rinsed and incubated with 100 μls of a PBS solution containinga 1:5 dilution of conditioned medium from each cell clone for 2 hours.Plates are then washed 3 times with PBS^(−/−) and incubated for 1 hourat room temperature with 50 μls of a PBS^(−/−) solution containing1:3000 dilution of a sheep anti-mouse horse radish peroxidase (HRP)conjugated secondary antibody such as anti-human IgG antibody. Platesare then washed 3 times with PBS^(−/−) and incubated with 50 μls ofTMB-HRP substrate (BioRad) for 15 minutes at room temperature to detectamount of antibody produced by each clone. Reactions are stopped byadding 50 μls of 500 mM sodium bicarbonate and analyzed by OD at 415 nmusing a BioRad plate reader. Clones exhibiting an enhanced signal overbackground cells (control cells with vector alone; control cells notcontaining the dominant negative mismatch repair allele) are thenisolated and expanded into 10 ml cultures for additionalcharacterization and confirmation of ELISA data in triplicateexperiments. ELISAs are also performed on conditioned (CM) from the sameclones to measure total Ig production within the conditioned medium ofeach well. Clones that produce an increased ELISA signal and haveincreased antibody levels are then further analyzed for variants thatare substantially free of FR-α binding competitors. Clones that producehigher OD values as determined by ELISA are further analyzed at thegenetic level to confirm the absence of FR-α binding competitors henceyielding a stronger ELISA signal. Briefly, 100,000 cells are harvestedand extracted for RNA using the Triazol method as described above. RNAsare reverse transcribed using Superscript II as suggested by themanufacturer (Life Technology) and PCR amplified for the antigen bindingsites contained within the variable light and heavy chains.

PCR reactions using degenerate oligonucleotides are carried out at 94°C. for 30 sec, 52° C. for 1 mm, and 72° C. for 1 min for 35 cycles.Products are analyzed on agarose gels. Products of the expectedmolecular weights are purified from the gels by Gene Clean (Bio 101),cloned into T-tailed vectors, and sequenced to identify the sequence ofthe variable light and heavy chains. Once the wild type sequence hasbeen determined, nondegenerate primers are made for RT-PCR amplificationof positive clones. Both the light and heavy chains are amplified, gelpurified and sequenced using the corresponding sense and antisenseprimers. The sequencing of RT-PCR products gives representative sequencedata of the endogenous immunoglobulin gene and not due to PCR-inducedmutations. Sequences from clones are then compared to the wild typesequence.

The methods of the invention yielded an anti-FR-α antibody comprising aheavy chain comprising an amino acid sequence of SEQ ID NO:5 and a lightchain comprising an amino acid sequence of SEQ ID NO:2. The molarextinction coefficient (ε) of the antibody was determined to be 43,320by measurement of the asorbance at 280 nm of 7.41 mg.ml solution ofantibody in 20 mM potassium phosphate, 150 mM NaCl at pH 7.2.

A single major band of Mr˜135 kD was observed upon separation of theantibody in SDS-PAGE under nonreducing conditions. Two bands of Mr˜55 kDand Mr˜25 kD were observed upon reduction. Purity was ascertained bydensitometric analysis of colloidal Coomassie blue-stained gels andfound to be greater than about 99.5% under reducing conditions andgreater than about 99% under nonreducing conditions.

Western blot analysis demonstrated that, when used to probe polypeptidesseparated on a nonreducing gel, the antibody was able to detect a singlepolypeptide of Mr˜35 kD in lysates prepared from a cell line known toexpress FR-α but not in lysates of a cell line that does not express theantigen (1205 Lu). The antibody also was able to detect soluble FR-αsecreted from KB cells, even after treatment of the antigen with PNGaseF to remove N-linked oligosaccharides.

Kinetic and steady-state binding constants between the antibody of theinvention and purified FR-α were determined by surface plasmon resonancespectroscopy. On-rate (k_(a)) was determined to be (2.25±0.02) M⁻¹s⁻¹,and off-rate (k_(d)) was determined to be (5.02±0.08) s⁻¹. A steadystate dissociation constant (K_(D)) of 2.23 nM was calculated.

Example 3 Binding of Antibody to Multimeric FR-α

Binding of a monoclonal antibody to the tetrameric form of FR-α wasshown by Western blot. Briefly, SK-Ov-3 and IGROV tumor cells were grownin nude mice and excised. Tumor tissues were lysed in RIPA buffer with15-20 strokes in a 2 ml Dounce tissue homogenizer. Insoluble materialwas removed by centrifugation and the total protein of the supernate wasdetermined using a BioRad protein Assay. In different experiments,either 5 ug or 20 ug of protein was run on a 4-12% Bis-Tris gel (MES)under non-reducing conditions. The electrophoresed protein wastransferred to a PVDF membrane. The membrane was blocked in Blotto (5%milk, 0.05% TBS-T). A 1:100 dilution of culture supernate from LK26hybridoma cells and total concentration of 0.1% NaN₃ was added directlyto the Blotto blocking solution as the primary antibody, and themembrane was incubated overnight. The membrane was washed in 0.05% TBS-Tand the secondary antibody (horseradish peroxidase labeled goat α-mouseIgG (heavy and light chains)) in Blotto blocking solution was added. Themembrane was developed using Super Signal West Pico ECL reagent. Theresults are shown in FIG. 1 (lane 1, SK-Ov-3; lane 2, IGROV). Theresults indicate that certain tumors that overexpress FR-α favor theproduction of multimeric FR-α over monomeric FR-α. This finding can beexploited by monoclonal antibodies that specifically recognize thetetrameric form of FR-α for the destruction of tumor tissue, whileleaving normal tissue (which generally expresses the monomeric form ofFR-α) unscathed.

Example 4 Expression of FR-α in Escherichia coli

Expression of FR-α was also assessed in Escherichia coli. Briefly, aplasmid containing the coding sequence for FR-α with a histidine tag(pBAD-His-hFR-α) was transfected into E. coli cells. A culture of E.coli containing plasmid pBAD-His-h FR-α was grown to OD₆₀₀=1.0.Thereafter, arabinose was added to a final concentration of 0.2%, andsamples were taken at the time points indicated in FIG. 2. E. colilysates were prepared by adding 25 ml of 4× LDS sample buffer to 65 mlculture. JAR cells were propagated in RPMI1640 medium containing 10%FBS, L-glutamine, sodium pyruvate, non-essential amino acids andpenicillin/streptomycin. The medium was removed from the cells and RIPAbuffer was added directly to the culture plates to lyse the cells forJAR cell extract controls. Samples were separated on a 4-12% NuPAGE gel(MES) and transferred to a PVDF membrane. After overnight blocking inTBST+5% milk, the membrane was probed with 1:1000 dilution of mAb LK26for 1 hr followed by a 1:10000 dilution of secondary antibody (goatα-mouse Ig conjugated to horseradish peroxidase) for 1 hr. Detection ofthe antibody was performed with Pierce Super Signal femto after anexposure of 5 minutes. The results are shown in FIG. 2 (lane 1, E.coli+pBAD-His-hFRa, induced 180 min.; lane 2, E. coli+pBAD-His-hFRa,induced 90 min.; lane 3, E. coli+pBAD-His-hFRa, induced 60 min.; lane 4,E. coli+pBAD-His-hFRa, induced 30 min.; lane 5, E. coli+pBAD-His-hFRa,induced 15 min.; lane 6, E. coli+pBAD-His-hFRa, uninduced; lane 7, JARcell extract).

Example 5 Multimeric Form of FR-α not an Artifact of Sample Preparation

To demonstrate that the multimeric FR-α was not an artifact ofaggregation in Triton X-100 micelles as described by Holm et al. (1997)Biosci. Reports 17(4):415-427, extracts of tumors were diluted in either1× RIPA (1% Triton X-100, 0.1% SDS, 180 mM NaCl, 20 mM potassiumphosphate, pH=7.2) or 1× PBS (150 mM NaCl, 20 mM potassium phosphate,pH=7.2). For all samples, 1 ug/ul of stock IGROV extract was used. Afterdilution, 4× LDS sample buffer was added to each sample to a finalconcentration of 1×. The samples were loaded on a 4-12% Bis-Tris gel inMES running buffer. Following electrophoresis, the protein wastransferred to a PVDF membrane. The membrane containing the transferredprotein was blocked for 48 hrs at room temperature in Blotto (5% skimmilk, 1× TBS, 0.05% Tween-20). The membrane was developed by incubatingthe membrane with a primary antibody (1 ug/ml LK26 antibody) followed bywashing, then incubation with a secondary antibody (HRP-conjugated goata-mouse IgG in Blotto). Following another washing step, the membrane wasdeveloped using a Super Signal West Pico ECL reagent and exposed for 1minute. The results are shown in FIG. 3 (lane 1, 1:100 dilution in PBS;lane 2, 1:50 dilution in PBS; lane 3, 1:25 dilution in PBS; lane 4, 1:10dilution in PBS; lane 5, 1:100 dilution in RIPA; lane 6, 1:25 dilutionin RIPA; lane 7, 1:10 dilution in RIPA; M, molecular weight markers,lane 8, 1:1 dilution in RIPA). Arrows indicate monomer (1×) and tetramer(4×). No treatment disrupted the tetrameric form of FR-α. The resultsindicate that certain tumors that overexpress FR-α express a multimericform of FR-α that has only been shown previously as artifacts of gelfiltration sample preparation.

Example 6 Screening Cells for ADCC Activity

The mAb-producing cells expressing the hPMS-134 will be subcloned byliming dilution and seeded in a flat-bottom 96-well plate. Seedingdensity will be determined empirically in order to obtain 40 single-cellcolonies per plate to approximate monoclonality.

The clones will be allowed to grow for a number of days, which will beempirically determined, after which a sufficient amount of antibody,capable of mediating ADCC activity, is produced. At the end of theincubation period, 50 ul of conditioned medium from each clone/well willbe used to assess concentration of antibodies by ELISA, while another 50ul of conditioned medium from the same well/clone will be utilized inthe ADCC assay. Briefly, for example, an anti-ovarian cancer mAb is usedin conjunction with the target cells, SKOV3 (passage 1 to 20, obtainedfrom ATCC), which are seeded the day before the assay in a flat-bottom96-well microplate at a density of 30,000 cell/well in complete growthmedium (RPMI-1640 containing 10% FBS, 2 mM L-glutamine). The followingday, the complete medium is replaced with 100 ul of CHO-CD serum-freemedium and 50 ul of antibody-containing conditioned medium will be addedto target cells and incubated for 20 minutes at 37° C. Subsequently, 100ul of serum-free medium containing 2×10⁵ effector cells are added toeach well and cells are incubated for 5-6 hours at 37° C., 5% CO₂.Plates are then briefly centrifuged and 100 ul of supernatant iscollected from each well and transferred into ELISA plates (Nunc). Onehundred ul of LDH substrate (Roche) is added to supernatants andincubated for 10 minutes at ambient temperature. LDH activity will beproportional to the extent of the LDH enzyme released from lysed targetcells. Optical density at 490 um (OD₄₉₀) is obtainedspectrophotometrically and percent of cytotoxicity is determined withthe formula: (sample OD₄₉₀−spontaneous OD₄₉₀)/(max OD₄₉₀−spontaneousOD₄₉₀)×100%, where ‘spontaneous’=target cells' lysis in absence ofeffector cells or antibody, and ‘max’=target cells' lysis in thepresence of 2% Triton. Cytotoxicity elicited by 100 ng/ml of a referenceantibody (protein A purified, parental antibody) will be used aspositive control. Non-specific cytotoxicity will be monitored using 100mg/ml of normal human IgG1. The ratio obtained by dividing the %cytotoxicity by the concentration of the antibody for each well/clone(i.e., ratio=50(%)/100(ng/ml)=0.5) will be set as the criterion forselecting lead clones. Lead clones will be expanded to 50 ml culturesand antibody will be purified from their conditioned media by protein-Aaffinity column as described. ADCC activities of the antibodies producedby the lead clones will be compared to the parental antibody usingconcentrations ranging from 10-1000 ng/ml.

In an alternative ADCC assay, the ability of antibody to produce ADCCwas evaluated using SKOV-3, IGROV-1, and 1205 Lu (negative control) astarget cells, and PBMCs from normal blood donors. Antibody was tested ata concentration of 10 micrograms/milliliter. Donor PBMCs used aseffector cells were thawed and kept overnight in medium (IMDMsupplemented with 10% FCS). The cells were resuspended in medium at aconcentration of 10⁷ cells/milliliter. The tumor cells used as targetcells were detached from the culture flask and 10⁶ cells in 100microliters FCS were labeled with 100 uCi (3.7 MBq) ⁵¹Cr (Amersham,Buckinghamshire, UK) for 2 hours at 37° C. Cells were washed thrice with5 milliliters medium and resuspended in medium at a concentration of 10⁵cells/milliliter. Fifty microliters of the tumor cells were seeded in Vbottom 96-well plates. Cells were then incubated with 50 microlitersmedium containing the test antibody or control antibody. After 30minutes incubation at 37° C., 50 microliters of the PBMCs were seeded inV bottom 96 well plates at various target-effector cell ratios (1:0,1:25, 1:50, and 1:100) and the plates were further incubated for 18hoursat 37° C. The release of ⁵¹Cr in the supernatant was determined in a LKBgamma-counter. Each measurement was carried out in triplicate. Thepercentage of release was defined as:

% release=[(release−spontaneous release)/(maximal release−spontaneousrelease)]×100.

The percentage of specific release was defined as:

% specific ⁵¹Cr release=% total ⁵¹Cr release with antibody−% total ⁵¹Crrelease without antibody.

Results:

TABLE 1 SKOV-3 Percentage of ⁵¹Cr release Patient 1 Patient 2 WithoutControl With Without Control With T:E ratio Antibody IgG Antibodyantibody IgG Antibody 1:0 1.3 ± 0.0 1.6 ± 0.0  2.0 ± 0.0 −1.4 ± 0.0 −0.7 ± 0.0  −0.6 ± 0.0 1:25 5.3 ± 0.3 5.0 ± 0.1 36.1 ± 1.4 2.6 ± 0.0 3.3± 0.0 31.2 ± 1.0 1:50 6.8 ± 0.1 5.9 ± 0.1 46.2 ± 1.0 4.5 ± 0.1 6.7 ± 0.143.5 ± 1.3 1:100 8.0 ± 0.2 8.3 ± 0.3 61.7 ± 0.2 7.6 ± 0.5 6.3 ± 0.8 56.0± 1.0

TABLE 2 SKOV-3 Percentage of specific ⁵¹Cr release Patient 1 Patient 2T:E ratio Control IgG Antibody Control IgG Antibody 1:0  0.3 ± 0.0  0.7± 0.0 0.7 ± 0.0  0.8 ± 0.0 1:25 −0.3 ± 0.4 30.8 ± 1.7 0.7 ± 0.1 28.6 ±1.0 1:50 −0.9 ± 0.2 39.4 ± 1.1 2.2 ± 0.2 39.0 ± 1.4 1:100  0.3 ± 0.353.7 ± 0.3 −1.3 ± 1.2  48.4 ± 1.5

TABLE 3 IGROV-I Percentage of ⁵¹Cr release Patient 1 Patient 2 WithoutControl With Without Control With T:E ratio Antibody IgG Antibodyantibody IgG Antibody 1:0 −3.0 ± 0.1 −4.9 ± 0.2 −4.1 ± 0.1 −13.3 ± 0.3 −12.0 ± 0.5  −10.9 ± 0.2  1:25 14.9 ± 3.3 20.0 ± 1.0 70.2 ± 1.3 15.6 ±2.9 13.4 ± 1.6 46.0 ± 1.2 1:50 15.2 ± 2.2 29.4 ± 2.3 66.8 ± 7.1 23.0 ±0.6 26.7 ± 0.5 64.7 ± 1.3 1:100 24.0 ± 4.1 33.8 ± 2.7 65.2 ± 1.2 36.8 ±2.4 41.1 ± 1.6  67.8 ± 10.5

TABLE 4 IGROV-I Percentage of specific ⁵¹Cr release Patient 1 Patient 2T:E ratio Control IgG Antibody Control IgG Antibody 1:0 −1.9 ± 0.3 −1.1± 0.2 1.3 ± 0.7 2.4 ± 0.5 1:25  5.1 ± 4.3 55.3 ± 4.4 −2.2 ± 4.4  30.4 ±4.1  1:50 14.2 ± 4.5 51.6 ± 9.3 3.7 ± 1.1 41.7 ± 1.9  1:100  9.8 ± 6.841.2 ± 5.3 4.3 ± 4.0 31.0 ± 12.9

ADCC assays using human ovarian cancer cells as target and peripheralblood mononuclear cells (PBMCs) as effector cells showed that anti-FR-αantibody mediated killing of tumor cell line SKOV-3. IGROV-1 aggregatedvery quickly and tended to form cell clumps. The cell line was sensitiveto killing by PBMCs alone. Control antibody also mediated some killing.Antibody mediated killing of IGROV-1.

Example 7 Immunohistochemistry Assay Using Anti-FR-α Antibody

Tissue preparation. Human tissue samples were obtained at autopsy orbiopsy. Tissues tested included adrenal, blood cells (granulocytes,lymphocytes, monocytes, platelets), blood vessels (endothelium), bonemarrow, brain (cerebrum (cortex), cerebellum), breast (mammary gland),eye, gastrointestinal tract (colon (large intestine), esophagus, smallintestine, stomach), heart, kidney (glomerulus, tubule), liver, lung,lymph node, ovary and fallopian tube (oviduct), pancreas, parathyroid,peripheral nerve, pituitary, placenta, prostate, salivary gland, skin,spinal cord, spleen, striated (skeletal) muscle, testis, thymus,thyroid, tonsil, ureter, urinary bladder, uterus (body (endometrium),cervix), ovarian carcinoma (carcinoma cells), ovarian carcinoma (stromalfibroblasts). Fresh unfixed tissue samples were placed in molds andfrozen on dry ice in TISSUE-TEK O.C.T. embedding medium. Tissue sampleswere sectioned and fixed for 10 minutes in room temperature acetone.Tissues were stored below −70° C. until staining. Just prior tostaining, the slides were fixed in 10% neutral buffered formalin.

Antibody preparation. Antibody was applied to tissue samples at twoconcentrations: 1 microgram/milliliter and 25 micrograms/milliliter.

Assays lacking primary antibody were used as an assay control. Mouseanti-fluorescein was used as secondary antibody. Goat anti-mouse IgG(GAMIgG)-peroxidase polymer was used as tertiary antibody.3,3′-diaminobenzidinen (DAB) was used as substrate chromogen.

Immunohistochemistry analysis. An indirect immunoperoxidase procedurewas performed. Acetone/formalin-fixed cryosections were rinsed twice inphosphate buffered saline (PBS [0.3M NaCl, pH 7.2]). Endogenousperoxidase was blocked by incubating the slides with peroxidase solutionof Dako EnVision Kit for 5 minutes followed by two rinses in phosphatebuffered saline. Slides were then treated with a protein block(phosphate buffered saline, 0.5% casein, 5% human gamma globulins, and 1mg/ml heat aggregated HuIgG (prepared by heating a 5 mg/ml solution to63° C. for 20 minutes and then cooling to room temperature)) designed toreduce nonspecific binding for 20 minutes. Following the protein block,primary antibody (anti-FR-α antibody, negative control antibody (HuIgG1or MsIgG1), or none) was applied at room temperature for one hour.Unconjugated secondary antibody (mouse anti-fluorescein) was applied for30 minutes. Slides were twice rinsed with PBS, treated withperoxidase-labeled goat anti-mouse IgG polymer (Dako EnVision kit) for30 minutes, rinsed twice with PBS, and treated with substrate-chromogen(DAB; Dako EnVision) for 8 minutes. Slides were rinsed in water,counterstained with hematoxylin, dehydrated, and coverslipped.

Results. The anti-FR-α antibody specifically and intensely stained humanovarian carcinoma cells (HT162) at both antibody concentrations as apositive control. Anti-FR-α antibody did not react with ovariancarcinoma (stromal fibroblasts) (negative control). Negative controlantibodies HuIgG1 and MsIgG1 did not specifically react with thepositive or negative control cells. No reactivity was observed with anytissues when primary antibody was omitted from the staining reaction.See Table 1.

Tissue cross-reactivity of anti-FR-α antibody.

TABLE 5 Cancer-specific expression of target antigen Tumor tissueExpression % positive samples Total number of origin by IHC of totaltested samples tested (n) Normal adult − 0 62 Ovarian +++++ 91 136carcinoma cells Breast ++++ 21 53 Renal ++++ 50 18 Colorectal +++ 22 27Lung +++ 33 18 Endometrial +++ 91 11 Brain +++ 80 5 Melanoma − 0 8lymphoma − 0 32 +/− indicates level of expression as detected byimmunohistochemistry

The antibodies of the invention do not react with stromal fibroblasts ofovarian carcinoma tissue (data not shown). Similar results forimmunohistochemical and tissue distribution analyses were obtained withthe antibodies of the invention in cynomolgus monkey and human (data notshown). Positive binding is seen in the cynomolgus monkey kidney cortex(proximal tubules and collecting ducts) and epithelium, tubular(membrane, cytoplasm/cytoplasmic granules), and uctules (membrane,cytoplasm) (data not shown).

In normal human tissues, anti-FR-α antibody specific staining wasobserved in tubular epithelium (kidney), bronchiolar epithelium (lung);pneumocytes (lung); epithelium of fallopian tube; and duct and ductileepithelium of the pancreas at both antibody concentrations.

In neoplastic human tissues, anti-FR-α antibody specific staining wasobserved in ovarian carcinoma tissue, endometrial carcinoma tissue, andrenal carcinoma tissue. Staining of ovarian and renal carcinoma cellsoccurred at the membrane and cytoplasm (data not shown).

These results are consistent with distribution of FR-α reported inliterature (Weitman, et al., Cancer Res., 61:3869-3876 (2001)).

In summary, FR-α is a glycoprotein whose expression is highly restrictedin normal tissues and highly expressed in a large portion of ovariantumors. Anti-FR-α antibodies of the invention are capable of inducingADCC, thus making the antibodies of the invention excellent drugcandidates for the treatment of a variety of cancers, including ovariancancer.

Example 8 Receptor Binding Activity

One of the major modes of action of unconjugated therapeutic monoclonalantibodies directed against tumor antigens is through recruitment ofimmune effector populations to the tumor cells (Clynes R, Takechi Y,Moroi Y, Houghton A, Ravetch J V. Proc. Natl. Acad. Sci. U.S.A. 1998Jan. 20; 95(2):652-6; Clynes R A, Towers T L, Presta L G, Ravetch J V.Nat. Med. 2000 April; 6(4):443-6). It is presumed that the efficiencywith which a given antibody can recruit immune effector cells to a tumorcell is influenced by the affinity of the antibody for its cognateantigen on the tumor cell surface, such that a high affinity antibodywill display more efficient recruitment of immune effectors to the tumorcell than a lower affinity counterpart recognizing the same antigen.Limited reports have attempted to demonstrate this relation in vitro(Alsmadi, 0. and Tilley, S A. J. Virol. 1998 January; 72(1):286-293;McCall, A M., Shahied, L., Amoroso, A R., Horak, E M., Simmons, R H.,Nielson, U., Adams, G P., Schier, R., Marks, J D., Weiner, L M. J.Immunol. 2001 May 15; 166(10):6112-7, as well as in vivo (Velders, M P,van Rhijn, C M., Oskam, G J., Warnaar, S O. and Litvinov, S V. J. Cancer1998; 78(4):476-483). In order to determine if such a correlationexists, in vitro ADCC activity of anti-FR-α antibodies and the affinityof these antibodies may be compared for their relevant antigen bysurface plasmon resonance spectroscopy.

Surface plasmon resonance spectroscopy relies on the short range (˜150nm) interaction of the electrical field (evanescent wave) generated byphotons under conditions of total internal reflection (TIR) withelectrons (surface plasmons) in a conductive film at the boundarybetween two media of differing refractive indices, whereby one of themedia is a thin gold layer (conductive film) coated with an alkanelinker coupled to CM-dextran. The CM-dextran surface, which forms anextended hydrogel in solution, projecting roughly 100-150 nm into theflowcell, may be derivatized further with a ligand of choice by covalentimmobilization to the carboxyl groups present on the CM-dextran layer.The angle necessary to allow the evanescent wave to interact with thegold layer will depend on the angle necessary to observe TIR, which inturn depends on the thickness or mass at the surface of the chip. Theinstrument thus allows for observation of the change in mass at thesurface of the chip over time, as would be observed when an analytewhich interacts with the immobilized ligand is injected into theflowcell. If injection of analyte is followed by injection of buffer,one can follow both the association (during injection of the analyte)and dissociation phases (during buffer injection) of the binding.Kinetic on-rates (k_(a)) and off-rates (k_(d)), as well as steady-stateequilibrium constants (K_(a) and K_(d)) can thus be extrapolated.

The soluble, secreted form of the antigen will be purified from theserum-free culture supernatant of target cells by chromatography throughPhenyl Sepharose (high sub), followed by ion exchange on S SepharoseFast Flow. Briefly, culture supernatant containing secreted antigen willbe loaded onto the Phenyl Sepharose (high sub) column in the absence ofadditional salts. Unbound proteins will be removed by extensive washingin HIC A (20 mM K phosphate pH 7.2), followed by elution of boundantigen using a linear gradient of 0-20 mM CHAPS in HIC buffer. Peakanti-FR-α antibody-containing fractions will be pooled, acidified (pH5.5) with 1 M citrate, then applied to a S Sepharose cation exchangecolumn. After washing with IEX buffer (20 mM K phosphate, pH 5.5), boundantigen will be eluted using a linear gradient of 0-1 M NaCl in IEXbuffer. Peak fractions will be pooled, concentrated using a Centriconcentrifugal concentration device (Millipore), and dialyzed against PBS.Based on the purity of the antigen preparation, an additional affinitychromatography step on covalently coupled folate Sepharose resin may benecessary (Sadasivan, E., da Costa, M., Rothenberg, S P. and Brink, L.Biochim. Biophys. Acta 1987; (925):36-47).

The antibody to be assayed will be purified in one step by affinitychromatography on recombinant protein A Sepharose resin (RPA-Sepharose,Amersham Biosciences) Immunoglobulin (Ig) containing tissue culturesupernatants will be loaded onto RPA-Sepharose columns by gravity, at anIg/ml resin value of 10 mg/mL of resin. Unbound proteins will be removedby extensive washing with PBS, followed by elution using 0.1 Mglycine-HCl pH 2.6. Fractions will be neutralized with 1 M Tris. Peakfractions will be pooled, and dialyzed against 1000 volumes of PBS. Igconcentration will be determined by BCA protein assay (Pierce ChemicalCo.) and Ig-specific ELISA.

Purified antigen will be diluted into coupling buffer (10 mM NaOAc pH5.0), and immobilized onto the flowcell of a CM5 sensor chip (Biacore)by amine coupling, using a mixture of N-hydroxysuccinimide (NHS) and1-ethyl-3-[dimethylaminopropyl]carbodiimide hydrochloride (EDC) toactivate carboxyl groups in the CM-Dextran hydrogel attached to thesurface of the CM5 sensor chip. Activated, underivatized carboxyl groupswill be quenched with 1 M ethanolamine A reference flowcell, consistingof the quenched CMDextran surface, activated in the absence of antigen,will be used to normalize all measurements. Crude, mAb-containingculture supernatants, or purified mAb preparations will be injected atflow rates of 30 ul/min for kinetic assays, and 5 ul/mm for steady-stateaffinity ranking experiments, using HBS-EP (20 mM HEPES-OH, 150 mM NaCl,3 mM EDTA, 0.005% Surfactant P-20, pH 7.4) as running buffer. PurifiedmAb preparations will be dialyzed against HBS-EP, using 10K MWCOSlide-A-Lyzer dialysis cassettes (Pierce) prior to their use in Biacoreanalysis. For samples containing tissue culture supernatant, BSA andsoluble CM-Dextran will be added to final concentrations of 1% and 1mg/ml, respectively. Regeneration of the surface will be accomplished by30 second injection of 50 mM NaOH, at a flow rate of 100 ul/min. Dataanalysis will be performed using Bia Evaluation software (Biacore).Kinetic data will be fitted to a simple 1:1 (Langmuir) binding model.For ranking experiments, rank will be determined by K_(D) valuesobtained from plots of Req versus C at different concentrations ofsample.

Example 9 Evaluation of Antibody in Human Tumor Xenograft Model

The SKOV-3 tumor cell line has been shown to express FR-α both on cellsin culture and in tumor xenografts. Antibody may be evaluated in vivousing the tumor xenograft model of SKOV-3 cells in mice. Paclitaxel maybe used as a positive control. Negative controls may be isotype matched,nonspecific murine IgG and vehicle control. Inhibition of tumor growthby the antibody relative to the negative controls is an indication thatthe antibody is useful in the treatment of ovarian cancer. The antibodypreferably demonstrates tumor growth inhibition of at least about 58%.

Example 10 Growth Inhibition Experiments

The sulforhodamine B (SRB) test (Shekan et al. (1990) J. Nat. CancerInst. 82:107-112, as modified by Keepers et al. (1991) Eur. J. Cancer27:897-900) may be used to test the effect of antibody treatment on thesusceptibility of cancer cells to treatment with antifolate compounds.Briefly (as described in Backus et al. (2000) Int. J. Cancer 87:771-778,cells are seeded in 100 ul medium (suitable for use with each particularcell line chosen for testing) in 96-well flat-bottom plates (intriplicate). Seeding density may vary according to the cell type used,but may be, for example, 8,000 cells/well for colon cancer cells,15,000cells/well for squamous cell carcinoma cells of the head and neck. Thecells are cultured in the presence of 1-100 ug/ml anti-folate receptorantibody. After 24 hours, 100 ul of drug containing medium is added andcells are cultured for an additional 72 hours. The concentration ofdrugs such as 5-fluoro-2′-deoxy-uridine-5′-monophosphate (FdUMP),leucovorin, ZD1649, MTA, GW1843U89, ZD9331, AG337, and PT523 ranges from1×10⁻⁵ to 1×10⁻¹¹ M. 5-FU is tested in a range of 1×10⁻⁴ to 1×10⁻¹⁰ Mwith or without 10 uM leucovorin. After 72 hrs of exposure to drug(s),the cells are fixed with trichloroacetic acid (TCA) and stained with SRBprotein dye. Results are expressed as % of control growth based on thedifference in optical density (OD₅₄₀) at the beginning and end of thedrug exposure period according to the formula published by Peters et al.((1993) Int. J. Cancer 54:450-455):

[(OD_(treated)/OD_(start of exposure))−1/[(OD_(control)/OD_(start of exposure))−1]×100%.

IC₅₀ values are calculated based on absorption values defined as drugconcentration corresponding to a reduction of cellular growth by 50%when compared with values of untreated control cells.

Example 11 Combination of Antifolate Antibodies and Antifolate Compounds

For combination therapy, efficacy may be demonstrated in vitro using theassay described above for ovarian cancer cell lines and the monoclonalantibodies of the invention. One of skill in the art may extrapolatedosages from the in vitro efficacy assays to determine a range ofefficacy in patients. Furthermore, dosages of antibodies accepted in theart for administration can be matched with dosages accepted for variousfolate inhibitors and adjusted to achieve maximum benefit with theminimum dosage. One of skill in the art is able to adjust these dosagesto achieve the desired effect with routine experimentation particularlywith the guidance on dosage for antibodies provided above and the assaydescribed for determining an effect in vitro.

What is claimed:
 1. A method of inhibiting the growth of folatereceptor-alpha-bearing cancer cells in a subject in need thereof, saidmethod comprising administering to said subject a therapeuticallyeffective amount of a composition comprising antibodies thatspecifically bind folate receptor-alpha, wherein each of said antibodiescomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:4 and a light chain comprising the amino acid sequence of SEQ ID NO: 1,and wherein said composition is free of a polypeptide consisting ofamino acids 20 to 111 of SEQ ID NO:24.
 2. The method of claim 1 whereinsaid heavy chain comprises the amino acid sequence of SEQ ID NO:
 5. 3.The method of claim 1 wherein said light chain comprises the amino acidsequence of SEQ ID NO:
 2. 4. The method of claim 1 wherein said heavychain comprises the amino acid of SEQ ID NO: 5 and said light chaincomprises the amino acid sequence of SEQ ID NO:
 2. 5. The method ofclaim 1 wherein said subject is a human subject.
 6. The method of claim1 wherein said antibodies are conjugated to a cytotoxic agent.
 7. Themethod of claim 1 wherein said subject is further administered at leastone antifolate compound.
 8. The method of claim 1 wherein said folatereceptor-alpha-bearing cancer cells are ovarian cancer cells, breastcancer cells, renal cancer cells, colorectal cancer cells, lung cancercells, endometrial cancer cells, brain cancer cells, fallopian tubecancer cells, uterine cancer cells, or leukemia cells.
 9. The method ofclaim 1 wherein said folate receptor-alpha-bearing cancer cells areepithelial ovarian cancer cells.
 10. The method of claim 1 wherein saidfolate receptor-alpha-bearing cells are cisplatin-resistant.
 11. Themethod of claim 1 wherein said composition comprises a homogeneouspopulation of antibodies.
 12. The method of claim 1 wherein each of saidantibodies further comprises a constant region.
 13. The method of claim1 wherein said composition further comprises a pharmaceuticallyacceptable carrier.
 14. The method of claim 1, wherein saidadministering comprises injecting or infusing said composition ofantibodies.
 15. A method for treating folate receptor-alpha-bearingcancer in a subject in need thereof, said method comprisingadministering to said subject a therapeutically effective amount of acomposition of antibodies that specifically bind folate receptor-alpha,wherein each of said antibodies comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 4 and a light chain comprising theamino acid sequence of SEQ ID NO: 1, and wherein said composition isfree of a polypeptide consisting of amino acids 20 to 111 of SEQ ID NO:24.
 16. The method of claim 15 wherein said heavy chain comprises theamino acid sequence of SEQ ID NO: 5 and said light chain comprises theamino acid sequence of SEQ ID NO:
 2. 17. The method of claim 15 whereinsaid cancer is epithelial ovarian cancer.
 18. The method of claim 15wherein said cancer is cisplatin-resistant.
 19. The method of claim 15wherein said subject is human.
 20. The method of claim 15 wherein saidadministering comprises injecting or infusing said composition ofantibodies.
 21. The method of claim 15 wherein said heavy chaincomprises the amino acid sequence of SEQ ID NO:
 5. 22. The method ofclaim 15 wherein said light chain comprises the amino acid sequence ofSEQ ID NO:
 2. 23. The method of claim 15 wherein said antibodies areconjugated to a cytotoxic agent.
 24. The method of claim 15 wherein saidsubject is further administered at least one antifolate compound. 25.The method of claim 15 wherein said folate receptor-alpha-bearing canceris ovarian cancer, breast cancer, renal cancer, colorectal cancer, lungcancer, endometrial cancer, brain cancer, fallopian tube cancer, uterinecancer, or leukemia.
 26. The method of claim 15 wherein said compositioncomprises a homogeneous population of antibodies.
 27. The method ofclaim 15 wherein each of said antibodies further comprises a constantregion.
 28. The method of claim 15 wherein said composition furthercomprises a pharmaceutically acceptable carrier.
 29. A method fortreating folate receptor-alpha-bearing cancer in a subject in needthereof, said method comprising administering to said subject atherapeutically effective amount of a composition of antibodies thatspecifically bind folate receptor-alpha, wherein each of said antibodiescomprises a heavy chain expressed from a cDNA comprising a nucleotidesequence encoding an amino acid sequence comprising SEQ ID NO: 4 and alight chain expressed from a cDNA comprising a nucleotide sequenceencoding an amino acid sequence comprising SEQ ID NO: 1, and whereinsaid composition is free of a polypeptide consisting of amino acids 20to 111 of SEQ ID NO:
 24. 30. The method of claim 29 wherein said heavychain is expressed from a cDNA comprising a nucleotide sequence encodingan amino acid sequence comprising SEQ ID NO:
 5. 31. The method of claim29 wherein said light chain is expressed from a cDNA comprising anucleotide sequence encoding an amino acid sequence comprising SEQ IDNO:
 2. 32. The method of claim 29 wherein said heavy chain is expressedfrom a cDNA comprising a nucleotide sequence encoding an amino acidsequence comprising SEQ ID NO: 5 and said light chain is expressed froma cDNA comprising a nucleotide sequence encoding an amino acid sequencecomprising SEQ ID NO:
 2. 33. The method of claim 29 wherein said heavychain is expressed from a cDNA comprising the nucleotide sequence of SEQID NO:
 7. 34. The method of claim 29 wherein said light chain isexpressed from a cDNA comprising the nucleotide sequence of SEQ ID NO:8.
 35. The method of claim 29 wherein said cancer is epithelial ovariancancer.
 36. The method of claim 29 wherein said cancer iscisplatin-resistant.
 37. The method of claim 29 wherein said subject ishuman.
 38. The method of claim 29 wherein said administering comprisesinjecting or infusing said composition of antibodies.
 39. The method ofclaim 29 wherein said antibodies are conjugated to a cytotoxic agent.40. The method of claim 29 wherein said subject is further administeredat least one antifolate compound.
 41. The method of claim 29 said folatereceptor-alpha-bearing cancer is ovarian cancer, breast cancer, renalcancer, colorectal cancer, lung cancer, endometrial cancer, braincancer, fallopian tube cancer, uterine cancer, or leukemia.
 42. Themethod of claim 29 wherein said composition comprises a homogeneouspopulation of antibodies.
 43. The method of claim 29 wherein each ofsaid antibodies further comprises a constant region.
 44. The method ofclaim 29 wherein said composition further comprises a pharmaceuticallyacceptable carrier.
 45. A composition of antibodies that specificallybind folate receptor-alpha, wherein each of said antibodies comprises aheavy chain expressed from a cDNA comprising a nucleotide sequenceencoding an amino acid sequence comprising SEQ ID NO: 4 and a lightchain expressed from a cDNA comprising a nucleotide sequence encoding anamino acid sequence comprising SEQ ID NO: 1, and wherein saidcomposition is free of a polypeptide consisting of amino acids 20 to 111of SEQ ID NO:
 24. 46. The composition of claim 45 wherein said heavychain is expressed from a cDNA comprising a nucleotide sequence encodingan amino acid sequence comprising SEQ ID NO:
 5. 47. The composition ofclaim 45 wherein said light chain is expressed from a cDNA comprising anucleotide sequence encoding an amino acid sequence comprising SEQ IDNO:
 2. 48. The composition of claim 45 wherein said heavy chain isexpressed from a cDNA comprising a nucleotide sequence encoding an aminoacid sequence comprising SEQ ID NO: 5 and said light chain is expressedfrom a cDNA comprising a nucleotide sequence encoding an amino acidsequence comprising SEQ ID NO:
 2. 49. The composition of claim 45wherein said heavy chain is expressed from a cDNA comprising thenucleotide sequence of SEQ ID NO:
 7. 50. The composition of claim 45wherein said light chain is expressed from a cDNA comprising thenucleotide sequence of SEQ ID NO:
 8. 51. The composition of claim 45wherein said antibodies are conjugated to a cytotoxic agent.
 52. Thecomposition of claim 45 comprising a homogeneous population ofantibodies.
 53. The composition of claim 45 wherein each of saidantibodies further comprises a constant region.
 54. The composition ofclaim 45 further comprising a pharmaceutically acceptable carrier.
 55. Acomposition of antibodies that specifically bind folate receptor-alpha,wherein each of said antibodies comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 4 and a light chain comprising theamino acid sequence of SEQ ID NO: 1, and wherein said composition isfree of a polypeptide consisting of amino acids 20 to 111 of SEQ ID NO:24.
 56. The composition of claim 55 wherein said heavy chain comprisesthe amino acid sequence of SEQ ID NO:
 5. 56. The composition of claim 55wherein said light chain comprises the amino acid sequence of SEQ ID NO:2.
 58. The composition of claim 55 wherein said heavy chain comprisesthe amino acid of SEQ ID NO: 5 and said light chain comprises the aminoacid sequence of SEQ ID NO:
 2. 59. The composition of claim 55comprising a homogeneous population of antibodies.
 60. The compositionof claim 55 wherein each of said antibodies further comprises a constantregion.
 61. The composition of claim 55 further comprising apharmaceutically acceptable carrier.
 62. A recombinant cell expressingantibodies that specifically bind folate receptor-alpha, wherein each ofsaid antibodies comprises a heavy chain expressed from a cDNA comprisinga nucleotide sequence encoding an amino acid sequence comprising SEQ IDNO: 4 and a light chain expressed from a cDNA comprising a nucleotidesequence encoding an amino acid sequence comprising SEQ ID NO: 1, andwherein said cell is free of a polypeptide consisting of amino acids 20to 111 of SEQ ID NO:
 24. 63. The cell of claim 62 wherein said heavychain is expressed from a cDNA comprising a nucleotide sequence encodingan amino acid sequence comprising SEQ ID NO:
 5. 64. The cell of claim 62wherein said light chain is expressed from a cDNA comprising anucleotide sequence encoding an amino acid sequence comprising SEQ IDNO:
 2. 65. The cell of claim 62 wherein said heavy chain is expressedfrom a cDNA comprising a nucleotide sequence encoding an amino acidsequence comprising SEQ ID NO: 5 and said light chain is expressed froma cDNA comprising a nucleotide sequence encoding an amino acid sequencecomprising SEQ ID NO:
 2. 66. The cell of claim 62 wherein said heavychain is expressed from a cDNA comprising the nucleotide sequence of SEQID NO:
 7. 67. The cell of claim 62 wherein said light chain is expressedfrom a cDNA comprising the nucleotide sequence of SEQ ID NO:
 8. 68. Thecell of claim 62 wherein said cell is free of a polynucleotide encodingan amino acid sequence consisting of amino acids 20 to 111 of SEQ ID NO:24.
 69. The cell of claim 62, wherein said cell expresses a homogeneouspopulation of antibodies.
 70. The cell of claim 62, wherein each of saidantibodies further comprises a constant region.
 71. A recombinant cellexpressing antibodies that specifically bind folate receptor-alpha,wherein each of said antibodies comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 4 and a light chain comprising theamino acid sequence of SEQ ID NO: 1, and wherein said cell is free of apolypeptide consisting of amino acids 20 to 111 of SEQ ID NO:
 24. 72.The cell of claim 71 wherein said heavy chain comprises the amino acidsequence of SEQ ID NO:
 5. 73. The cell of claim 71 wherein said lightchain comprises the amino acid sequence of SEQ ID NO:
 2. 74. The cell ofclaim 71 wherein said heavy chain comprises the amino acid of SEQ ID NO:5 and said light chain comprises the amino acid sequence of SEQ ID NO:2.
 75. The cell of claim 71 wherein said cell is free of apolynucleotide encoding an amino acid sequence consisting of amino acids20 to 111 of SEQ ID NO:
 24. 76. The cell of claim 71, wherein said cellexpresses a homogeneous population of antibodies.
 77. The cell of claim71, wherein each of said antibodies further comprises a constant region.78. A method of generating a composition of antibodies that specificallybind folate receptor-alpha, said method comprising recombinantlyexpressing in a cell a cDNA comprising a nucleotide sequence encoding aheavy chain comprising the amino acid sequence of SEQ ID NO: 4 and acDNA comprising a nucleotide sequence encoding a light chain comprisingthe amino acid sequence of SEQ ID NO: 1, and recovering a composition ofantibodies expressed by said cell, wherein each of said antibodiescomprises said heavy chain and said light chain, and wherein saidcomposition of antibodies is free of a polypeptide consisting of aminoacids 20 to 111 of SEQ ID NO:
 24. 79. The method of claim 78 whereinsaid heavy chain comprises the amino acid sequence of SEQ ID NO:
 5. 80.The method of claim 78 wherein said light chain comprises the amino acidsequence of SEQ ID NO:
 2. 81. The method of claim 78 wherein said heavychain comprises the amino acid sequence of SEQ ID NO: 5 and said lightchain comprises the amino acid sequence of SEQ ID NO:
 2. 82. The methodof claim 78 wherein said cDNA comprising a nucleotide sequence encodinga heavy chain comprises the nucleotide sequence of SEQ ID NO:
 7. 83. Themethod of claim 78 wherein said cDNA comprising a nucleotide sequenceencoding a light chain comprises the nucleotide sequence of SEQ ID NO:8.
 84. The method of claim 78 wherein said cell is free of apolynucleotide encoding an amino acid sequence consisting of amino acids20 to 111 of SEQ ID NO:
 24. 85. The method of claim 78, wherein saidcell expresses a homogeneous population of antibodies.
 86. The method ofclaim 78, wherein each of said antibodies further comprises a constantregion.